Establishment of cancer cell lines from rat hepatocholangiocarcinoma and assessment of the role of granulocyte-colony stimulating factor and hepatocyte growth factor in their growth, motility and survival.

BACKGROUND/AIMS: Oval cells (OCs), putative hepatic stem cells, may give rise to liver cancers. We developed a carcinogenesis regimen, based upon induction of OC proliferation prior to carcinogen exposure. In our model, rats subjected to 2-acetylaminofluorene/ partial-hepatectomy followed by aflatoxin injection (APA regimen) developed well-differentiated hepatocholangiocarcinomas. The aim of this study was to establish and characterize cancer cell lines from this animal model. METHODS: Cancer cells were cultured from animals sacrificed eight months after treatment, and single clones were selected. The established cell lines, named LCSCs, were characterized, and their tumorigenicity was assessed in vivo. The roles of granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) in LCSC growth, survival and motility were also investigated. RESULTS: From primary tumors, six cell lines were developed. LCSCs shared with the primary tumors the expression of various OC-associated markers, including cMet and G-CSF receptor. In vitro, HGF conferred protection from death by serum withdrawal. Stimulation with G-CSF increased LCSC growth and motility, while the blockage of its receptor inhibited LCSC proliferation and migration. CONCLUSIONS: Six cancer cell lines were established from our model of hepatocholangiocarcinoma. HGF modulated LCSC resistance to apoptosis, while G-CSF acted on LCSCs as a proliferative and chemotactic agent.

Isolation and characterization of hepatic stem cells, or "oval cells," from rat livers.

The pace of research on the potential therapeutic uses of liver stem cells or "oval cells" has accelerated significantly in recent years. Concurrent advancements in techniques for the isolation and characterization of these cells have helped fuel this research. Several models now exist for the induction of oval cell proliferation in rodents. Protocols for the isolation and culture of these cells have evolved to the point that they may be set up in any laboratory equipped for cell culture. The advent of magnetic cell sorting has eliminated reliance on expensive flow cytometric sorting equipment to generate highly enriched populations of oval cells. Our laboratory has had much success in using the oval cell surface marker Thy-1 in combination with magnetic sorting to produce material suitable for testing the influence of a myriad of chemical signaling molecules on the oval cell phenotype. This chapter will describe our basic strategy for oval cell induction and isolation. Additionally, two in vitro procedures are described which the reader may find useful in the early stages of developing an oval cell research project.

Granulocyte-colony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats.

BACKGROUND AND AIMS: Hepatic regeneration is a heterogeneous phenomenon involving several cell populations. Oval cells are considered liver stem cells, a portion of which derive from bone marrow (BM). Recent studies have shown that granulocyte-colony stimulating factor (G-CSF) may be effective in facilitating liver repair. However, it remains unclear if G-CSF acts by mobilizing BM cells, or if it acts locally within the liver microenvironment to facilitate the endogenous restoration program. In the present study, we assessed the involvement of G-CSF during oval cell activation. METHODS: Dipeptidyl-peptidase-IV-deficient female rats received BM transplants from wild-type male donors. Four weeks later, rats were subjected to the 2-acetylaminofluorene/partial hepatectomy model of oval cell-mediated liver regeneration, followed by administration of either nonpegylated G-CSF or pegylated G-CSF. Control animals did not receive further treatments after surgery. The magnitude of oval cell reaction, the entity of BM contribution to liver repopulation, as well as the G-CSF/G-CSF-receptor expression levels were evaluated. In addition, in vitro proliferation and migration assays were performed on freshly isolated oval cells. RESULTS: Oval cells were found to express G-CSF receptor and G-CSF was produced within the regenerating liver. G-CSF administration significantly increased both the magnitude of the oval cell reaction, and the contribution of BM to liver repair. Finally, G-CSF acted as a chemoattractant and a mitogen for oval cells in vitro. CONCLUSIONS: We have shown that G-CSF facilitates hepatic regeneration by increasing the migration of BM-derived progenitors to the liver, as well as enhancing the endogenous oval cell reaction.

Bone marrow-derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration.

BACKGROUND AND AIMS: The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). METHODS: Three groups of mutant F344 dipeptidyl peptidase IV-deficient (DPPIV(-)) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV(+)) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. RESULTS: In group A, DPPIV(+) hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and alpha-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. CONCLUSIONS: Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.