Differentiation Of Hepatoblasts Research Articles

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.

Oncostatin M Renders Epithelial Cell Adhesion Molecule–Positive Liver Cancer Stem Cells Sensitive to 5-Fluorouracil by Inducing Hepatocytic Differentiation

Recent evidence suggests that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells; CSC). Although normal stem cells and CSCs are considered to share similar self-renewal programs, it remains unclear whether differentiation programs are also maintained in CSCs and effectively used for tumor eradication. In this study, we investigated the effect of oncostatin M (OSM), an interleukin 6-related cytokine known to induce the differentiation of hepatoblasts into hepatocytes, on liver CSCs. OSM receptor expression was detected in the majority of epithelial cell adhesion molecule-positive (EpCAM(+)) HCC with stem/progenitor cell features. OSM treatment resulted in the induction of hepatocytic differentiation of EpCAM(+) HCC cells by inducing signal transducer and activator of transcription 3 activation, as determined by a decrease in stemness-related gene expression, a decrease in EpCAM, alpha-fetoprotein and cytokeratin 19 protein expressions, and an increase in albumin protein expression. OSM-treated EpCAM(+) HCC cells showed enhanced cell proliferation with expansion of the EpCAM-negative non-CSC population. Noticeably, combination of OSM treatment with the chemotherapeutic agent 5-fluorouracil (5-FU), which eradicates EpCAM-negative non-CSCs, dramatically increased the number of apoptotic cells in vitro and suppressed tumor growth in vivo compared with either saline control, OSM, or 5-FU treatment alone. Taken together, our data suggest that OSM could be effectively used for the differentiation and active cell division of dormant EpCAM(+) liver CSCs, and the combination of OSM and conventional chemotherapy with 5-FU efficiently eliminates HCC by targeting both CSCs and non-CSCs.

Abstract 4281: Oncostatin M renders epithelial cell adhesion molecule-positive liver cancer stem cells sensitive to 5-fluorouracil by inducing hepatocytic differentiation

Abstract Recent evidence suggests that a certain type of hepatocellular carcinoma (HCC) is hierarchically organized by a subset of cells with stem cell features (cancer stem cells: CSCs). Although normal stem cells and cancer stem cells (CSCs) are considered to share similar self-renewal programs, it is uncertain whether differentiation signaling is kept maintained in both types of cells. In this study, we investigated the effect of oncostatin M (OSM), an interleukin 6-related cytokine known to induce the differentiation of hepatoblasts into hepatocytes, on liver CSCs. Recombinant human OSM was used for both in vitro and in vivo experiments. Epithelial cell adhesion molecule (EpCAM) was used for the isolation of liver CSCs. Gene and protein expression was evaluated by quantitative reverse transcription-PCR, immunohistochemistry, and intracellular fluorescence-activated cell sorting. Apoptosis was evaluated by Annexin V and activated-caspase 3 staining. Tumor initiating capability was examined by the subcutaneous tumor formation in NOD/SCID mice. OSM receptor expression was detected in the majority of EpCAM-positive (EpCAM+) HCC with stem/progenitor cell features. OSM treatment (100 ng/ml) resulted in the induction of hepatocytic differentiation of EpCAM+ liver CSCs derived from hepatocellular carcinoma (HCC) cell lines and primary HCCs, in terms of decrease of alpha-fetoprotein and cytokeratin 19 production and increase of albumin production. Furthermore, OSM treated cells exhibited reduced expression of stemness-related genes including Bmi1 and POU5F1. While pretreatment of HCC cell lines with OSM before transplantation in vitro resulted in the decreased tumorigenic capacity, the OSM treatment after transplantation in vivo showed small tumor suppressing effects irrespective of hepatocytic differentiation of the tumor. Intriguingly, when the OSM treatment was combined with chemotherapeutic reagent 5-fluorouracil to eradicate non-CSCs, we observed the dramatic increase of apoptotic cells in vitro and the suppression of tumorigenesis in vivo compared with either OSM or 5-fluorouracil treatment alone. In conclusion, our data suggest that OSM can be effectively utilized for the differentiation and active cell division of dormant EpCAM+ liver CSCs, and the combination of OSM and conventional chemotherapy such as 5-fluorouracil efficiently eliminates HCC by targeting both CSCs and non-CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4281.

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog ( Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk + hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk + hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk + hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.

Notch2 signaling promotes biliary epithelial cell fate specification and tubulogenesis during bile duct development in mice

Intrahepatic bile duct (IHBD) development begins with the differentiation of hepatoblasts into a single continuous biliary epithelial cell (BEC) layer, called the ductal plate. During ductal plate remodeling, tubular structures arise at distinct sites of the ductal plate, forming bile ducts that dilate into the biliary tree. Alagille syndrome patients, who suffer from bile duct paucity, carry Jagged1 and Notch2 mutations, indicating that Notch2 signaling is important for IHBD development. To clarify the role of Notch2 in BEC differentiation, tubulogenesis, and BEC survival, we developed a mouse model for conditional expression of activated Notch2 in the liver. We show that expression of the intracellular domain of Notch2 (Notch2ICD) differentiates hepatoblasts into BECs, which form additional bile ducts in periportal regions and ectopic ducts in lobular regions. Additional ducts in periportal regions are maintained into adulthood and connect to the biliary tight junction network, resulting in an increased number of bile ducts per portal tract. Remarkably, Notch2ICD-expressing ductal plate remnants were not eliminated during postnatal development, implicating Notch2 signaling in BEC survival. Ectopic ducts in lobular regions did not persist into adulthood, indicating that local signals in the portal environment are important for maintaining bile ducts. Notch2 signaling regulates BEC differentiation, the induction of tubulogenesis during IHBD development, and BEC survival.

Notch signaling in bile duct development: New insights raise new questions

Notch signaling in bile duct development: New insights raise new questions

New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts

Hepatoblasts have the potential to differentiate into both hepatocytes and biliary epithelial cells through a differentiation program that has not been fully elucidated. With the aim to better define the mechanism of differentiation of hepatoblasts, we isolated hepatoblasts and established new culture systems. We isolated hepatoblasts from E12.5 fetal mouse liver by using E-cadherin. The E-cadherin+ cells expressed alpha-fetoprotein (AFP) and albumin (Alb) but not cytokeratin 19 (CK19). Transplantation of the E-cadherin+ cells into mice that had been subjected to liver injury or biliary epithelial injury led to differentiation of the cells into hepatocytes or biliary epithelial cells, respectively. In a low-cell-density culture system in the absence of additional growth factors, E-cadherin+ cells formed colonies of various sizes, largely comprising Alb-positive cells. Supplementation of the culture medium with hepatocyte growth factor and epidermal growth factor promoted proliferation of the cells. Thus the low-cell-density culture system should be useful to identify inductive factors that regulate the proliferation and differentiation of hepatoblasts. In a high-cell-density system in the presence of oncostatin M+dexamethasone, E14.5, but not E12.5, E-cadherin+ cells differentiated into mature hepatocytes, suggesting that unidentified factors are involved in hepatic maturation. Culture of E-cadherin+ cells derived from E12.5 or E14.5 liver under high-cell-density conditions should allow elucidation of the mechanism of hepatic differentiation in greater detail. These new culture systems should be of use to identify growth factors that induce hepatoblasts to proliferate or differentiate into hepatocytes and biliary epithelial cells.

Transcriptional profiling implicates TGFβ/BMP and Notch signaling pathways in ductular differentiation of fetal murine hepatoblasts

Bile duct morphogenesis involves sequential induction of biliary specific gene expression, bilayer generation, cell proliferation, remodeling and apoptosis. HBC-3 cells are a model system to study differentiation of hepatoblasts along the hepatocytic or bile ductular lineage in vitro and in vivo. We used microarray to define molecular pathways during ductular differentiation in response to Matrigel. The temporal pattern of expression of marker genes induced was similar to that observed during bile duct formation in vivo. Notch, HNF1β, Polycystic kidney disease 2, Bicaudal C 1 and β-catenin were up regulated during the time course. Functional clustering analysis revealed significant up regulation of clusters of genes involved in extracellular matrix remodeling, ion transport, vacuoles, lytic vacuoles, pro-apoptotic and anti-apoptotic genes, transcription factors and negative regulators of the cell proliferation, while genes involved in the cell cycle were significantly down regulated. Notch signaling pathway was activated by treatment with Matrigel. In addition, TGFβ/BMP signaling pathway members including the type I TGFβ receptor and Smads 3, 4 and 5 were significantly up regulated, as were several TGFβ/BMP responsive genes including Hey 1, a regulator of Notch pathway signaling. SMADS 3, 4 and 5 were present in the nuclear fraction of HBC-3 cells during ductular differentiation in vitro, but not during hepatocyte differentiation. SMAD 5 was preferentially expressed in hepatoblasts undergoing bile duct morphogenesis in the fetal liver, while the TGFβ/BMP signaling antagonist chordin, was expressed throughout the liver suggesting a mechanism by which TGFβ/BMP signaling is limited to hepatoblasts that contact portal mesenchyme in vivo.

Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.

Differential Expression of the Rat γ-Glutamyl Transpeptidase Gene Promoters along with Differentiation of Hepatoblasts into Biliary or Hepatocytic Lineage

gamma-Glutamyl transpeptidase (GGT), a major enzyme of glutathione (GSH) homeostasis, is often used as a biliary marker to follow the differentiation of hepatic precursor cells. The expression of the GGT gene is driven by different promoters and yields multiple mRNAs, depending on the cell type or the stage of differentiation. In the present study, we analyzed the GGT mRNA expression pattern by quantitative reverse transcriptase-polymerase chain reaction or by in situ hybridization i) in the liver, in vivo, at early stages of development; ii) in oval cells, which proliferate and differentiate into hepatocytes in response to galactosamine injury in vivo; and finally, iii) during hepatoblast differentiation, in vitro. We show that GGT gene transcription originates from promoters P3, P4, and P5 in rat hepatic precursor cells. Differentiation of these cells induces profound alterations in GGT gene expression, leading to extinction of promoters P4 and P5, when they differentiate into the hepatocytic pathway, and to extinction of promoters P3 and P5 when they differentiate into the biliary pathway. This diversity in GGT mRNA expression provides unique molecular probes to follow hepatic precursor cell differentiation. Furthermore, the identification of factors governing GGT P5 and P4 promoter expression should provide further insight into the molecular events that occur as the liver precursor cell differentiates into the hepatic lineages.

Hepatocytes Deficient in CCAAT/Enhancer Binding Protein α (C/EBPα) Exhibit both Hepatocyte and Biliary Epithelial Cell Character

To further elucidate the role of CCAAT/Enhancer Binding Protein α (C/EBPα) in hepatocyte differentiation, we investigated fetal and newborn C/EBPα-deficient (C/EBPα −/−) mice using confocal microscopy and markers specific for hepatocyte (AFP) and biliary epithelial cell (A6) differentiation. Histologically, in fetal liver of C/EBPα −/− mice, pseudoglandular structures appeared starting at 16.5 days of gestation. In newborn livers, the diameters of these structures greatly increased. They were randomly distributed between portal and central veins and interfered with the establishment of normal hepatic plates. However, the portal bile ducts developed normally. The pseudoglandular structures were lined with small hepatocytes with round nuclei and were positive for both AFP and A6 antigens. These data show that C/EBPα −/− hepatocytes exhibit biliary epithelial cell characters and suggest an involvement of C/EBPα in the control of the switch in the differentiation of bi-potential hepatoblasts along the hepatocyte lineage.