Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.     

Direct In Vivo Cell Lineage Analysis in the Retrorsine and 2AAF Models of Liver Injury after Genetic Labeling in Adult and Newborn Rats

Backgrounds and Aims

When hepatocyte proliferation is impaired, liver regeneration proceeds from the division of non parenchymal hepatocyte progenitors. Oval cells and Small Hepatocyte-like Progenitor Cells (SHPCs) represent the two most studied examples of such epithelial cells with putative stem cell capacity. In the present study we wished to compare the origin of SHPCs proliferating after retrorsine administration to the one of oval cells observed after 2-Acetyl-Amino fluorene (2-AAF) treatment.

Methodology/Principal Findings

We used retroviral-mediated nlslacZ genetic labeling of dividing cells to study the fate of cells in the liver. Labeling was performed either in adult rats before treatment or in newborn animals. Labeled cells were identified and characterised by immunohistochemistry. In adult-labeled animals, labeling was restricted to mature hepatocytes. Retrorsine treatment did not modify the overall number of labeled cells in the liver whereas after 2-AAF administration unlabeled oval cells were recorded and the total number of labeled cells decreased significantly. When labeling was performed in newborn rats, results after retrorsine administration were identical to those obtained in adult-labeled rats. In contrast, in the 2-AAF regimen numerous labeled oval cells were present and were able to generate new labeled hepatocytes. Furthermore, we also observed labeled biliary tracts in 2-AAF treated rats.

Conclusions

Our results srongly suggest that SHPCs are derived from hepatocytes and we confirm that SHPCs and oval cells do not share the same origin. We also show that hepatic progenitors are labeled in newborn rats suggesting future directions for in vivo lineage studies.     

Coexpression of Stem Cell Factor andc-kitin Embryonic and Adult Liver

Stem cell factor and its receptorc-kitconstitute an important signal transduction system implicated in survival, proliferation, and differentiation of stem cells in hematopoiesis, gametogenesis, and melanogenesis. In the present study we used both immunocytochemical methods and Western analysis to demonstrate the presence of this cytokine/receptor system in both embryonic and adult rat liver. Stem cell factor was present in the ductular cells around the portal vein during the late embryonic stage of the liver. In the adult liver both bile ducts and bile ductules were positive for stem cell factor andc-kit.When the activation of the liver stem cell compartment was induced by combining administration of acetylaminofluorene and partial hepatectomy, both stem cell factor andc-kitwere expressed in the infiltrating oval cell population, but absent in the newly formed basophilic hepatocytes. Activation of oval cell proliferation following administration ofD-galactosamine also produced a similar but less prominent increase in the level of the stem cell factor. Our data suggest that the stem cell factor/c-kitsignal transduction system is involved in the development of bile ducts and that it may also be an important member of the growth factor/receptor systems associated with the biology of liver stem cells.     

Hepatic progenitor cells in human liver diseases

The canals of Hering and bile ductules in human liver contain hepatic progenitor cells that can differentiate towards the biliary and hepatocytic lineage. Proliferation and differentiation of hepatic progenitor cells is referred to as 'activation' and this process occurs to a variable degree in almost all human liver diseases. Several studies indicate that hepatic progenitor cell activation in diseased liver is regulated by neural and neuroendocrine factors such as the vagal innervation. Analogous to oval cells in animal liver, there is evidence that human hepatic progenitor cells may be able to give rise to hepatocellular carcinoma and other liver tumors.     

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (α-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

Developmental changes in myelin-induced proliferation of cultured Schwann cells

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.     

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.     

The stem-cell reserve of the liver]

This paper considers the problem of epithelial stem cells of the liver and possibilities of its experimental solution. Authors' own data obtained with the model of induced hepatocarcinogenesis in mice are discussed; the experiments were performed using electron microscopy, autoradiography and immunochemistry. In accordance with these data, Gering cells are stem cells of the liver, and oval cells correspond to committed precursors capable to differentiation in either hepatocellular or cholangiolar direction under the conditions of periportal microenvironment. We have also compared hepatocyte differentiation in preneoplastic mouse liver and regenerating pancreas of adult rats (Rao et al. Amer. J. Pathol. 1989. V. 134. P. 1069-1086). We also discuss stem cell compartment organization in organs having glandular structure and the possibility of existence in the adult of non-committed multipotent cells capable of producing various types of differentiation in tissues having common origin during embryogenesis.     

Germinal cells in the goldfish retina that produce rod photoreceptors

Dividing cells and their progeny in retinae of young goldfish were labeled with [3H]thymidine, and selected cells were reconstructed from serial sections processed for electron microscopic autoradiography. Our goals were to characterize the cells that were identified as rod precursors in previous light microscopic autoradiographical studies and to determine their origin and fate. (In fish the population of rods increases several-fold postembryonically by proliferation of rod precursor cells scattered across the retina). Over 200 labeled cells taken from 11 retinas were examined, and 20 of these were reconstructed in their entirety. Some retinas were examined at short intervals (1 to 48 hr) after [3H]thymidine injection in order to study mitotically active cells, and others were examined after longer intervals (9 or 14 days) to discover the nature of the progeny of labeled dividing cells. Previous evidence from thymidine studies in larval goldfish suggested that proliferating cells destined to produce rods appear first in the inner nuclear layer and later in the outer nuclear layer, where they continue to divide and generate new rods (P.R. Johns, (1982) J. Neurosci. 2, 179). The present results provide morphological evidence in support of the suggestion that rod precursors migrate from inner to outer nuclear layer and, furthermore, show that the precursors are closely associated with, and perhaps guided by, the radial processes of Müller glial cells. Examination of EM autoradiographs of labeled cells at 9 and 14 days after a pulse label with thymidine confirms that the differentiated progeny of dividing precursor cells are exclusively rods. To our knowledge, rod precursors are the first example of a neuronal germinal cell in the vertebrate central nervous system that under normal conditions produces only one type of neuron.     

Identification of putative bovine mammary epithelial stem cells by their retention of labeled DNA strands

Stem cells appear to retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily injections of 5-bromo-2-deoxyuridine (BrdU). Five weeks later, BrdU-labeled mammary epithelial cells were still evident. The percentage of BrdU-labeled epithelial cells was greatest in the lower region of the mammary gland, near the gland cistern, and was decreased toward the periphery of the parenchymal region, where the ducts were invading the mammary fat pad. Increased numbers of BrdU-labeled epithelial cells in basal regions of the gland are likely a consequence of decreased proliferation rates and increased cell cycle arrest in this area. In peripheral regions of mammary parenchyma, the percentage of heavily labeled epithelial cells averaged 0.24%, a number that is consistent with estimates of the frequency of stem cells in the mouse mammary gland. Epithelial label-retaining cells seemingly represent a slowly proliferating population of cells, as 5.4% of heavily labeled cells were positive for the nuclear proliferation antigen Ki67. Because epithelial label-retaining cells contain estrogen receptor (ER)-negative and ER-positive cells, they apparently comprise a mixed population, which I suggest is composed of ER-negative stem cells and ER-positive progenitors. Continuing studies will address the usefulness of this technique to identify bovine mammary stem cells and to facilitate studies of stem cell biology.     

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.     

Common antigens of oval cells and cholangiocytes in the mouse. Their detection by using monoclonal antibodies

Monoclonal antibodies (MAb) were produced against antigens (Ag) of oval cells isolated from the preneoplastic murine liver. To suppress the immune response to major antigens common with hepatocytes, the principle of anti-idiotype immunization was employed. Characteristics of three MAb reacting selectively with the foci of oval cell proliferation are described. MAb A6 and G7 detected two different antigens (Ag A6 and Ag G7, respectively) common for oval cells and cholangiocytes. Ag A6 was also found in normal parenchyma (in membranes of single hepatocytes adjacent to portal veins), in the preneoplastic liver (in hepatocytes formed de novo) and in some hepatoma cells. Ag G7 was not detected in hepatocytes. MAb E5 stained the matrix in the areas adjacent to oval cells and large bile ducts. All the three Ag were widely distributed in normal tissues of mice. The significance of the detected Ag as markers of murine liver epithelial cell lines and stages of their differentiation is discussed as well as the possible relationship between Ag A6 and Ag of human blood groups.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

Lymphocytes support oval cell-dependent liver regeneration

In case of hepatic damage, the liver uses a unique regeneration mechanism through proliferation of hepatocytes. If this process is inhibited, bipotent oval stem cells proliferate and differentiate to hepatocytes and bile ducts, thus restoring liver mass. Although oval cell accumulation in the liver is often associated with inflammatory processes, the role of lymphocytes in oval cell-mediated hepatic regeneration is poorly understood. We treated wild-type and immunodeficient mice with an oval cell-inducing diet: in the absence of T cells (CD3epsilon(-/-) and Rag2(-/-)) there were fewer oval cells, whereas in alymphoid mice (Rag2(-/-)gamma(c)(-/-)) a strongly reduced oval cell response and higher mortality, due to liver failure, was observed. Adoptive transfer of T cells into alymphoid mice protected them from liver failure, but was insufficient to restore the oval cell response. Treatment of Rag2(-/-) mice with an NK cell-depleting Ab resulted in a significantly diminished oval cell response. These genetic experiments point to a major role for NK and T cells in oval cell expansion. In wild-type mice, oval cell proliferation is accompanied by an intrahepatic inflammatory response, characterized by the recruitment of Kupffer, NK, NKT, and T cells. Under these conditions, lymphocytes produce T(H)1 proinflammatory cytokines (IFN-gamma and TNF-alpha) that are mitogenic for oval cells. Our data suggest that T and NK lymphocytes stimulate oval cell expansion by local cytokine secretion. This beneficial cross-talk between the immune system and liver stem cells operates under noninfectious conditions and could promote tissue regeneration following acute liver damage.     

Hepatic oval 'stem' cell in liver regeneration

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes and bile ductular cells. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Expression of the Hoxa-13 gene correlates to hepatitis B and C virus associated HCC

To study the Hoxa-13 gene in the liver, we examined its expression by RT-PCR in various liver cell lines, rat livers under different conditions, and human primary hepatocellular carcinomas (HCCs). The gene was found to be expressed in cell lines originating from liver stem-like cells, but not in cell lines originating from hepatocytes and bile duct epithelia. Expression was induced in rat livers after treatment with d-galactosamine, which is known to induce oval cell proliferation, but not after a two-thirds partial hepatectomy (2/3 PH) where induction of oval cell proliferation is thought not to occur. Expression of the gene correlated with human HCC samples associated with Hepatitis B or C virus infection in this small series. These results suggest that the Hoxa-13 gene may provide a potentially useful tool for elucidation of mechanisms involved in lineage-specific differentiation and carcinogenesis of liver stem cells.