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DEVELOPMENT AND DISEASE RESEARCH ARTICLE 1951 Development 136, 1951-1960 (2009) doi:10.1242/dev.031369 Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver Mayuko Okabe1,*, Yuko Tsukahara1,*, Minoru Tanaka1,*,†, Kaori Suzuki1, Shigeru Saito1, Yoshiko Kamiya1, Tohru Tsujimura2, Koji Nakamura3 and Atsushi Miyajima1 Hepatic oval cells are considered to be facultative hepatic stem cells (HSCs) that differentiate into hepatocytes and cholangiocytes in severely injured liver. Hepatic oval cells have also been implicated in tumorigenesis. However, their nature and origin remain elusive. To isolate and characterize mouse oval cells, we searched for cell surface molecules expressed on oval cells and analyzed their nature at the single-cell level by flow cytometric analysis and in the in vitro colony formation assay. We demonstrate that epithelial cell adhesion molecule (EpCAM) is expressed in both mouse normal cholangiocytes and oval cells, whereas its related protein, TROP2, is expressed exclusively in oval cells, establishing TROP2 as a novel marker to distinguish oval cells from normal cholangiocytes. EpCAM+ cells isolated from injured liver proliferate to form colonies in vitro, and the clonally expanded cells differentiate into hepatocytes and cholangiocytes, suggesting that the oval cell fraction contains potential HSCs. Interestingly, such cells with HSC characteristics exist among EpCAM+ cells of normal liver. Intriguingly, comparison of the colony formation of EpCAM+ cells in normal and injured liver reveals little difference in the number of potential HSCs, strongly suggesting that most proliferating mouse oval cells represent transit-amplifying cells rather than HSCs. INTRODUCTION Most of the metabolic functions in the liver are carried out by hepatocytes that form hepatic cords, whereas cholangiocytes form bile ducts that drain bile produced by hepatocytes. During development, hepatocytes and cholangiocytes, two types of hepatic epithelial cells, derive from hepatoblasts emerging from the foregut endoderm (Notenboom et al., 2003; Zaret, 2000). Hepatoblasts are highly proliferative and express hepatocytic proteins such as albumin (ALB) and alpha-fetoprotein (AFP), an immature hepatocyte marker. As the liver develops, hepatoblasts propagate and those close to the portal mesenchyme differentiate into cholangiocytes, while the rest become mature hepatocytes (Lemaigre, 2003). Therefore, hepatoblasts are thought to be hepatic stem/progenitor cells in the fetus. By contrast, it is highly controversial whether adult mammalian liver contains hepatic stem cells (HSCs). Adult liver has a remarkable potential to regenerate from severe parenchymal loss, even though hepatocytes and cholangiocytes are mitotically dormant under normal conditions. Hepatocytes have a remarkable potential to self-replicate (Fausto, 2004; Michalopoulos and DeFrances, 1997) and are capable of at least 80 doublings by serial transplantation (Overturf et al., 1997), allowing the liver to regenerate. However, liver injury that limits this pathway accompanies the proliferation of a potential stem/progenitor cell compartment at the interface of the biliary tree and hepatic cords, which is known as the ductular reaction (Alison et al., 1996; Roskams et al., 2004; Theise et al., 1999). These undifferentiated 1 Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. 2Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan. 3 LivTech, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan. *These authors contributed equally to this work Author for correspondence (e-mail: [email protected]) † Accepted 31 March 2009 epithelial cells are often referred to as 'oval cells' because of their ovoid nucleus (Farber, 1956). Upon activation, oval cells expand into liver parenchyma from the portal area. Oval cells express both ALB and cytokeratin 19 (CK19; KRT19 – Mouse Genome Informatics), which are hepatocytic and cholangiocytic markers, respectively, and are believed to differentiate into hepatocytic and biliary lineages, similar to hepatoblasts in embryonic liver. Thus, oval cells are thought to be facultative stem/progenitor cells in adult liver. Although oval cells have been most extensively studied in rodents, similar cells have been found in connection with various human liver diseases and are implicated in tumorigenesis (Fausto, 2004; Lee et al., 2006). Whether oval cells constitute HSCs has been debated in numerous reports involving various rodent injury models using chemical reagents, including carcinogenic agents. The 2acetylaminofluorene (2-AAF)/partial hepatectomy (PH) model, in which hepatocyte proliferation is blocked by 2-AFF prior to PH, has been extensively used to characterize oval cells in rats (Evarts et al., 1987; Laishes and Rolfe, 1981). However, the same procedure does not generate oval cells in mice and alternatives such as the use of a choline-deficient ethionine-supplemented diet (Akhurst et al., 2001) and a 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC)containing diet (Preisegger et al., 1999; Wang et al., 2003) have been developed. Although the proliferating epithelial cells that are present in the periportal region upon injury caused by various insults are referred to as oval cells, it remains unclear whether oval cells generated via different protocols in different species have the same characteristics. A major problem in characterizing oval cells is the lack of appropriate cell surface markers to identify and isolate the oval cell compartment. Therefore, despite a large number of studies, the exact nature of oval cells – including their origin, stemness and bi-directional differentiation – is still poorly understood. Because of the difficulty in performing clonal analysis of HSCs, it has also been unclear whether HSCs exist in normal liver. The aim of this study was to identify cell surface molecules on mouse oval cells and to analyze their nature at the clonal level. To this end, we utilized the 2-AAF/PH rat model and the DDC diet DEVELOPMENT KEY WORDS: Hepatic stem cell, Oval cell, EpCAM, TROP2 (TACSTD2), Hepatocyte, Cholangiocyte, Liver injury 1952 RESEARCH ARTICLE Development 136 (11) mouse model to generate oval cells and found that epithelial cell adhesion molecule (EpCAM) and the related molecule, TROP2 (TACSTD2), were upregulated in these livers. EpCAM was expressed in normal cholangiocytes and also in oval cells in the liver of mice fed the DDC diet (DDC liver). By contrast, TROP2 was expressed in almost all EpCAM+ cells in DDC liver, but not in normal liver, indicating that TROP2 is a novel marker to distinguish between normal cholangiocytes and oval cells. Furthermore, we isolated EpCAM+ cells from DDC liver and demonstrated that clonally expanded cells were able to differentiate into hepatocytes and cholangiocytes. Finally, we provide evidence for the presence of potential HSCs in EpCAM+ cells of normal liver and compare their characteristics before and after oval cell activation. was recovered and redigested with basic perfusion solution containing 0.5 g/l collagenase type IV, 0.5 g/l pronase (Roche Diagnostics) and 50 mg/l DNase I (Sigma) by stirring for 20 minutes at 37°C. This digested liver was also passed through a 70-μm cell strainer and the flow-through fraction was combined with the first cell suspension. After centrifugation at 700 rpm (100 g) for 2 minutes, the supernatant was transferred to a new tube and the centrifugation repeated until no cell pellet was visible. The final supernatant was centrifuged at 1200 rpm (300 g) for 5 minutes and the precipitated cells were used as non-parenchymal cells (NPCs) for FCM analysis. Aliquots of cells were blocked with anti-FcR antibody, co-stained with fluorescein- and biotin-conjugated antibodies, washed, incubated with allophycocyaminconjugated streptavidin (Invitrogen), and analyzed by FACSCalibur (Becton Dickinson). Dead cells were excluded by propidium iodide staining. MATERIALS AND METHODS Antibodies and immunohistochemistry (IHC) Total RNA was prepared from the non-parenchymal fraction of rat liver treated with 2-AAF/PH at 7 days after PH as described previously (Tanimizu et al., 2004b). Total RNA was amplified using the MessageAmp aRNA Amplification Kit (Ambion), and used to construct a cDNA library in the pMXs-SST vector using the SuperScript Choice System (Invitrogen, Carlsbad, CA, USA). The cDNA library contained 4.3⫻106 independent clones. The signal sequence trap method was performed as described previously (Kojima and Kitamura, 1999). The anti-EpCAM monoclonal antibody was generated by immunization of a rat with a Ba/F3 cell transfectant overexpressing EpCAM cDNA. The establishment of hybridoma clones was performed as described previously (Hara et al., 1999). The anti-EpCAM monoclonal antibody was biotinylated using Amersham ECL Protein Biotinylation Module (GE Healthcare, UK) or fluorescein-conjugated using the Fluorescein Labeling Kit-NH2 (Dojindo Molecular Technologies), and then used for all FCM analyses. The rabbit anti-mouse CK19 polyclonal antibody was raised against the C-terminal peptide, HYNNLPTPKAI, and the serum was used for IHC as previously described (Tanimizu et al., 2003). The biotin-conjugated anti-mouse TROP2 antibody was purchased from R&D Systems. The anti-human Ki67 antibody was purchased from Becton Dickinson. Frozen sections (8 μm) of livers were prepared using a HM505E cryostat (Microm International) after fixation with 4% paraformaldehyde, and incubated with each antibody, followed by a biotin- or fluorescein-conjugated secondary antibody. The signals were visualized by fluorescence microscopy. Preparation of liver cells and flow cytometry (FCM) RNA extraction and reverse transcription PCR (RT-PCR) A single-cell suspension from DDC and normal livers was obtained by a modified two-step collagenase perfusion method as described previously (Seglen, 1976). In short, livers were perfused with liver perfusion medium (Invitrogen) at a flow rate of 3 ml/minute for 5 minutes. Then, the liver was perfused with basic perfusion solution (136 mM NaCl, 5.4 mM KCl, 5 mM CaCl2, 0.5 mM NaH2PO3 2H2O, 0.42 mM Na2HPO3 12H2O, 10 mM HEPES pH 7.5, 5 mM glucose and 4.2 mM NaHCO3) containing 0.5 g/l collagenase type IV (Sigma, St Louis, MO, USA) at a flow rate of 3 ml/minute for 8 minutes. The digested liver was transferred to a glass dish and chopped into small pieces using a surgical knife in D-PBS (Dulbecco's phosphate-buffered saline). Cells dispersed by pipetting were passed through a 70-μm cell strainer and the flow-through fraction was used for the next step as the first cell suspension. The undigested clot on the strainer Total RNA was extracted from each cell preparation using Trizol reagent (Invitrogen). Total RNA (1 μg) and random hexamer primers were used to synthesize cDNA using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech). The samples were denatured at 94°C for 5 minutes, then subjected to 25-40 cycles of denaturation at 94°C for 30 seconds, annealing at 52-57°C for 30 seconds, and extension at 72°C for 1 minute, with the final extension at 72°C for 5 minutes. PCR primers for mouse genes are shown in Table 1. The quantitative real-time RT-PCR was performed using a LightCycler ST300 (Roche) and the following primers (5⬘ to 3⬘): rat Epcam, TCTACAAGGAAGAGATCAGCAAAA and TGTGTATCTCACCCATCTCCTTT; rat Trop2, GACCAAATGTGTTGGCCTGT and GTCACAGCTGGGAGGAAAAT; rat Hprt, GACCGGTTCTGTCATGTCG and ACCTGGTTCATCATCACTAATCAC. Animals C57BL/6 mice (Japan SLC, Hamamatsu, Japan) at 8-12 weeks were used for all experiments. All experiments with animals were performed according to institutional guidelines. The diet containing 0.1% DDC was purchased from CLEA, Japan. Mouse oval cells were activated by feeding with the diet containing 0.1% DDC. Identification of cDNA encoding a membrane protein Table 1. Oligonucleotides used in RT-PCR Epcam Ck19 Ck7 Alb Afp Gapdh Hprt G6Pase Tat Cps integrin β4 Ggt1 mucin 1 Trop2 claudin 4 Cd44 Slc10a1 Tdo2 Forward (5⬘ to 3⬘) CGGCTCAGAGAGACTGTGTC GTCCTACAGATTGACAATG GGGATGACCTCCGCAACACC CATGACACCATGCCTGCTGAT CTGGAGTGTCTGCAGGATGG GGAGCGAGACCCCACTAA GACTGAAAGACTTGCTCGAG AACCCATTGTGAGGCCAGAGG TGCATCCTCCTGAAGACATG ACTGAGAGATGCTGACCCTA GACCTATGAAGAAGGTGCTC GCTGAGCTGATTGAGCATCCG GAGCGCCAGCCTTGAGTTTG CTGACCTAGACTCCGAGCTG GACTTTGACCCCTGCAGAGG CAGAGGCGACTAGATCCCTC AGATCAAGGCTCACTTCTGG ACAATGAAGAAGACAGAGC Reverse (5⬘ to 3⬘) GATCCAGTAGGTCCTCACGC CACGCTCTGGATCTGTGACAG CTCCAGCAGCTTGCGGTAGG GCCTTTCCACCAGGGATCCAC CCACAGCCGGACCATTTCTC GTGTAGCCCAAGATGCCC CCAGCAAGCTTGCAACCTTAACAA TACTCATTACACTAGTTGGTC CTTCTCTCTGGTGTAGCTCT CCTGGAAATTGGTGAGGAGA GGCTCAGATGCGTGCCATAG GGTTGATGAAGTTGGGCGAGC GGAGGCACTACTGTGGACTG CCAACCCATCTGGTCTGAGG GGCCACAGGCTGTTATGAGC GAGTCACAGTGCGGGAACTC AGAAGTCCTTCTGCAAGCTG TGTAGTCTCCTCCAAAGTTA DEVELOPMENT Gene Hepatic stem cells in adult liver RESEARCH ARTICLE 1953 Culture of EpCAM+ cells and colony formation assay NPCs were prepared as described above. EpCAM+ cells were sorted by FACSVantage SE (Becton Dickinson). The cells were suspended in the standard medium (Williams' medium E containing 10% FBS, 10 mM nicotinamide, 2 mM L-glutamine, 0.2 mM ascorbic acid, 20 mM HEPES pH 7.5, 1 mM sodium pyruvate, 17.6 mM NaHCO3, 14 mM glucose, 100 nM dexamethasone, 1⫻ ITS (insulin, transferrin, selenium X) and 50 mg/ml gentamicin) and seeded on a type-I collagen-coated dish. human EGF, human recombinant HGF and mouse IL6 were added to the culture to a final concentration of 10 ng/ml each. After the establishment of cell lines, IL6 was excluded from the culture medium because it was confirmed to have no apparent effect. For colony formation assays, EpCAM+ cells were sorted by a two-step selection (see Fig. S1 in the supplementary material) and plated at 1⫻104 cells per 35-mm dish. The isolated cells were cultured for 9 days and then the number and size of colonies were counted. Differentiation into the hepatocytic lineage in vitro In vitro differentiation into the cholangiocytic lineage Cellmatrix Type I-A (Nitta Gelatin) was used for three-dimensional culture to induce cholangiocytic differentiation according to the manufacturer's instructions. In short, 0.3% Cellmatrix Type I-A, a 5⫻ DMEM containing 250 mg/ml gentamicin, and the reconstitution buffer (0.05 M NaOH, 200 mM HEPES pH 7.5, 262 mM NaHCO3) were mixed at a ratio of 7:2:1. This mixture was mixed with an equal volume of 5⫻104 cells suspended in standard culture medium without dexamethasone and nicotinamide but with human recombinant HGF (20 ng/ml). The cell suspension was poured into a 6-well plate and left at 37°C to form a gel. Then, the culture medium was gently laid onto the gel. RESULTS Screening for cell surface markers of oval cells To identify the cell surface molecules expressed on hepatic oval cells, we utilized the signal sequence trap (SST) method, which can efficiently isolate genes encoding a protein with a signal sequence (Kojima and Kitamura, 1999; Watanabe et al., 2007). As there are several protocols for generating oval cells in rats and mice, the characteristics of oval cells might not be the uniform. We therefore searched for cell surface molecules expressed on oval cells in the two species using different protocols (see Fig. S2 in the supplementary material). To this end, we first constructed an SST cDNA library from non-parenchymal cells (NPCs) of rat liver subjected to 2-AAF/PH treatment and identified 54 membrane proteins (see Table S1 in the supplementary material). First, we compared the expression of their counterpart genes for mouse between normal and DDC liver by RT-PCR, and found that Epcam, Trop2, mucin 1, claudin 4, Cd44, integrin β3, Lyve1, gp130 (Il6st) and Fxyd5 were significantly upregulated in DDC liver relative to normal liver. Chronic liver injury caused by DDC diet induces oval cells in mice, whereas PH and carbon tetrachloride-induced acute hepatitis do not. Next, the expression of the candidate genes was compared by northern blotting among normal liver, DDC liver and models of acute hepatitis, resulting in the identification of six of the nine genes that were specifically upregulated in the DDC liver, but not in the other livers (Fig. 1A). The remaining three genes, Lyve1, gp130 and Fxyd5, were upregulated in an acute hepatitis model, suggesting that they might be involved in inflammation (data not shown). Because the expression of EpCAM, mucin 1, CD44 and Fig. 1. Expression profiles of candidate genes in normal and injured mouse and rat liver. (A) Northern blot analysis of candidate genes in mouse liver. The expression of these genes was selectively upregulated in DDC liver, but not in injured liver without oval cell activation. (B) Quantitative RT-PCR of Epcam and Trop2 in rat liver. Whereas Epcam was expressed in normal rat liver (cont.) and upregulated in 2-AAF/partial hepatectomy (PH)-treated liver, Trop2 was not expressed in normal liver but was expressed in 2-AAF/PH-treated liver. N, adult mouse normal liver; O, DDC liver (6 weeks); P, liver 48 hours after 70% PH; C, liver 24 hours after carbon tetrachloride administration. claudin 4 has been reported to be upregulated in the rat oval cell fraction (Yovchev et al., 2007), the characteristics of mouse oval cells in the DDC model appear similar to those of rat oval cells in the 2-AAF/PH model. In this study, we have focused on two structurally related type-I membrane proteins, EpCAM and TROP2. EpCAM is known to be expressed in many types of normal epithelial cell as well as in tumor cells (Armstrong and Eck, 2003; Went et al., 2004). In the liver, EpCAM is expressed on cholangiocytes but not on hepatocytes (de Boer et al., 1999; Momburg et al., 1987). Consistent with previous studies (Yovchev et al., 2007; Yovchev et al., 2008), real-time PCR showed that Epcam was expressed in normal rat liver and its expression was upregulated by 2-AAF/PH (Fig. 1B). As for mouse oval cells, Gleiberman et al. reported that EpCAM is expressed in oval cells upon carbon tetrachloride-induced liver injury (Gleiberman et al., 2005). However, the expression status of EpCAM in DDC liver and the nature of isolated EpCAM+ cells have remained unknown. By contrast, Trop2 was expressed in both rat and mouse injured liver, but not in normal liver (Fig. 1). TROP2 is a member of the EpCAM family and exhibits nearly 50% homology with EpCAM. TROP2 has been shown to be expressed in various tumors, whereas its expression in the liver was not known. We further examined the expression of EpCAM and TROP2 on mouse oval cells in DDC liver and investigated the nature of isolated EpCAM+ cells. Expression of EpCAM in mouse hepatic oval cells As shown in Fig. 2A, after 4 weeks of DDC diet feeding, the mouse liver turned black because of hepatic porphyria resulting from the inhibition of the heme biosynthetic pathway (Fonia et al., 1996). Hematoxylin and Eosin (H&E) staining was performed in adult normal liver and DDC liver. The DDC liver clearly showed numerous small cells with a large nucleus around the portal veins (Fig. 2B). Immunohistochemistry (IHC) for CK19, a marker for oval cells and DEVELOPMENT Clonally expanded cells (3⫻105 per well) were cultured in the standard culture medium in a 6-well plate. After 2 days, 20 ng/ml Oncostatin M (OSM) and 1% DMSO were added into the confluent culture. After 5 days, the medium was changed to standard culture medium containing 20 ng/ml OSM, 1% DMSO and 17% Matrigel (growth factor reduced). After 3 and 5 days, the cultured cells were used for RNA preparation and periodic acidSchiff (PAS) staining as described (Kamiya et al., 1999). 1954 RESEARCH ARTICLE Development 136 (11) Fig. 2. DDC diet causes hepatic injury and oval cell activation. (A) The liver turned black after mice were fed a DDC diet. (B) H&E staining of a frozen section of mouse normal liver (top) and 4 weeks after DDC feeding (middle and bottom). Numerous small cells appeared around the portal veins in the DDC liver (arrows). The brown clots represent the deposition of iron hemes (arrowheads). (C) Immunohistochemistry (IHC) with antiCK19 antibody showed that these numerous small cells included CK19expressing oval cells (arrows) in DDC liver. PV, portal vein. Scale bars: 100 μm TROP2 is a novel marker for mouse oval cells Because TROP2 expression was specifically upregulated in both the mouse and rat injury models with oval cell activation (Fig. 1), TROP2 was anticipated to be expressed in oval cells. To reveal TROP2-expressing cells in normal and injured liver, we performed IHC with anti-EpCAM and anti-TROP2 antibodies. In contrast to EpCAM, TROP2 was not expressed in normal mouse liver (Fig. 4A). However, numerous TROP2+ cells appeared around the portal area in DDC liver (Fig. 4B). Double immunostaining of TROP2 and EpCAM clearly showed that most of the EpCAM+ cells coexpressed TROP2 in DDC liver, suggesting that TROP2 is a novel marker for oval cells (Fig. 4B). Although we could not distinguish the original bile duct in DDC liver, almost all EpCAM+ cells expressed TROP2. FCM of the NPCs prepared from DDC liver also showed that the expression level of TROP2 and the population of TROP2+ cells among EpCAM+ cells gradually increased upon ingestion of the DDC diet (Fig. 4C). Consistent with the result of IHC, whereas TROP2 was not present in cholangiocytes expressing EpCAM at day 0, almost all EpCAM+ cells became TROP2+ in the DDC-fed mice and EpCAM+ TROP2– cells were hardly detected after 4 weeks, suggesting that cholangiocytes themselves might also begin to express TROP2 by oval cell activation in the DDC model. Alternatively, it is also possible that TROP2+ oval cells differentiate into mature cholangiocytes and replace the pre-existing cholangiocytes damaged by DDC administration. Characterization of mouse oval cells To reveal the characteristics of mouse oval cells, the gene expression profile of freshly isolated EpCAM+ cells from DDC liver was examined by RT-PCR. As previously reported in rat oval cells, mouse EpCAM+ cells also expressed both cholangiocytic markers [Ck19, Ck7 (Krt7) and Ggt1] and a hepatocytic marker (Alb), whereas the other NPCs did not (Fig. 5A). Consistent with the previous report that AFP expression was rarely detected in mouse oval cells (Jelnes et al., 2007), AFP was not detected in mouse EpCAM+ cells (data not shown). Rat oval cells were reported to express c-KIT, CD34 and THY1 (Petersen et al., 1998). It was also reported that CD133 (PROM1) is expressed in both mouse and rat oval cells (Rountree et al., 2007; Suzuki et al., 2008; Yovchev et al., 2008). Taking advantage of FCM using the anti-EpCAM antibody, we investigated the expression of these oval cell markers in EpCAM+ cells before and after DDC feeding (Fig. 5B) and found DEVELOPMENT cholangiocytes, demonstrated that these cells included oval cells as well as other CK19-negative cells, such as inflammatory cells and fibroblasts (Fig. 2C). Since EpCAM expression was upregulated in DDC liver, it was examined by IHC using sections of normal liver and of liver from mice fed DDC for 1 or 4 weeks. EpCAM+ bile ducts were located adjacent to the portal vein in normal liver as reported previously (de Boer et al., 1999; Hreha et al., 1999; Joplin et al., 1990), whereas there were many EpCAM+ cells forming ductular structures away from the portal vein in DDC liver (Fig. 3A). IHC with both antiEpCAM and anti-CK19 antibodies demonstrated that all CK19+ cells expressed EpCAM in DDC liver (Fig. 3B). Thus, all oval cells expressing CK19 also expressed EpCAM. To further investigate the EpCAM+ cells, we generated rat monoclonal antibodies against mouse EpCAM that were applicable for flow cytometry (FCM). FCM of NPCs prepared from DDC liver showed that neither CD45 (PTPRC) nor PECAM was expressed on EpCAM+ cells, indicating that EpCAM+ cells are not hematopoietic or endothelial cells (Fig. 3C). Furthermore, the isolated EpCAM+ cells were individually examined by immunostaining after cytospin. Almost all the sorted cells were immunostained with anti-CK19 antibody as well as A6 antibody, a mouse oval cell marker (Engelhardt et al., 1993) (Fig. 3D). Hepatic oval cells are known to be highly proliferative. To investigate their proliferation in vivo, the isolated EpCAM+ cells were stained with anti-Ki67 antibody (Fig. 3E). Whereas the percentage of Ki67+ cells in the isolated EpCAM+ cells was ~1% in normal liver, it was 12.2% after 1 week on the DDC diet and increased to 17.4% after 4 weeks (Fig. 3F). These results strongly suggested that EpCAM is expressed in proliferating oval cells and that anti-EpCAM antibody is useful for isolating oval cells from mice fed with DDC. Hepatic stem cells in adult liver RESEARCH ARTICLE 1955 Fig. 3. EpCAM is a cell surface marker for mouse oval cells. (A) IHC of frozen liver sections with anti-EpCAM antibody after DDC feeding. EpCAM was expressed in cholangiocytes around the portal vein of normal mouse liver (0 w). Feeding DDC caused the proliferation of EpCAM+ cells (1 and 4 weeks). (B) IHC of frozen liver sections with anti-EpCAM and anti-CK19 antibodies after 4 weeks of DDC feeding. (C) Flow cytometry (FCM) of nonparenchymal cells (NPCs) prepared from the liver of mice fed DDC for 4 weeks with anti-EpCAM antibody and either CD45 or PECAM antibody. EpCAM+ cells were negative for CD45 (hematopoietic marker) and PECAM (endothelial marker). (D) Immunostaining of EpCAM+ cells with anti-A6 and anti-CK19 antibodies by cytospin. EpCAM+ cells expressed both molecules. (E) Immunostaining of EpCAM+ cells sorted from normal and DDC liver with anti-Ki67 antibody by cytospin. Many EpCAM+ cells from DDC liver were stained with Ki67 (arrowheads). (F) The percentage of Ki67+ cells among EpCAM+ cells after DDC treatment. The data are derived from five different fields of view. Error bars, s.d. PV, portal vein. Scale bars: 100 μm. Primary culture of EpCAM+ cells from DDC liver It has been thought that oval cells are proliferative in vivo and possess the potential to differentiate into hepatocytic and cholangiocytic lineages. Therefore, oval cells are considered facultative stem/progenitor cells. To assess such characteristics, we cultured the EpCAM+ cells isolated from DDC liver in vitro. The EpCAM+ cells purified with a cell sorter were seeded onto type-I collagen in the presence of EGF, HGF and IL6. After plating, adherent cells began to proliferate (Fig. 6A) and covered the entire dish after 1 month. The in vitro proliferating EpCAM+ cells were apparently homogeneous and exhibited epithelial cell morphology even after serial passages (Fig. 6B). They could grow and survive for more than 6 months, maintaining a homogenous morphology. These results suggested that EpCAM+ cells derived from DDC liver include cells with high proliferative potential. Fig. 4. Expression of EpCAM and TROP2 in normal and injured mouse liver. (A,B) IHC of frozen sections of normal liver (A) and the liver of mice fed DDC for 5 weeks (B) with anti-EpCAM and anti-TROP2 antibodies. TROP2 was expressed in oval cells but not in normal cholangiocytes. (C) FCM of NPCs with anti-EpCAM and antiTROP2 antibodies after DDC feeding. TROP2 begins to be expressed in EpCAM+ cells as DDC feeding proceeds. PV, portal vein. Scale bars: 100 μm. DEVELOPMENT that c-KIT, CD34 and THY1 were not expressed in EpCAM+ cells regardless of injury. The expression pattern of CD133 was similar to that of EpCAM, indicating that both molecules are originally expressed in normal cholangiocytes. By contrast, TROP2 was exclusively expressed in oval cells of DDC liver. These results indicate that TROP2 is induced by oval cell activation and that it is a novel marker for oval cells in mice. 1956 RESEARCH ARTICLE Development 136 (11) Fig. 5. Characterization of freshly isolated EpCAM+ cells. (A) RT-PCR of freshly isolated EpCAM+ and EpCAM– cells from the liver of mice fed DDC for 4 weeks. NPCs from DDC liver were divided into EpCAM+ and EpCAM– cells by FACSVantage, then RT-PCR was performed. (B) FCM of EpCAM+ cells from normal and DDC livers with known oval cell markers. EpCAM+ cells surrounded by bold lines were reanalyzed with other antibodies (TROP2, CD133, CD34, c-KIT, THY1) as shown. Differentiation of clonally expanded EpCAM+ cells into the hepatocytic lineage in vitro To address whether the established clones can differentiate into hepatocytic and cholangiocytic lineages, we used two clones, HSCE1 and HSCE2, and examined their differentiation potential. To induce hepatocytic differentiation and maturation, we utilized an in vitro culture system reported previously (Kamiya et al., 1999; Kamiya et al., 2002). Oncostatin M (OSM) is a powerful inducer of the differentiation of fetal hepatocytes and the addition of Engelbreth-Holm-Swarm (EHS) gel enhances hepatocyte maturation. Dimethyl sulfoxide (DMSO) is also known to maintain the differentiation of hepatocytes in culture (Isom et al., 1985; Sakai et al., 2002). By combining these methods, the expression of metabolic enzymes and transporters was monitored in two clones (Fig. 7A). Because both clones exhibited a similar profile, only the results of HSEC1 are shown. The expression of G6Pase, Cps, Tat, tryptophan-2,3-dioxygenase (Tdo2) and solute carrier family 10 (sodium/bile acid cotransporter family), member 1 (Slc10a1) was markedly induced by the addition of OSM, DMSO and EHS gel (Fig. 7B). By contrast, the expression of Ck19 and of Afp, a marker of immature fetal hepatocytes, was downregulated. In addition, the PAS reaction showed that there were many clusters of hepatocytic Fig. 6. EpCAM+ cells derived from DDC liver have high proliferative potential. (A,B) In vitro culture of EpCAM+ cells. Freshly isolated EpCAM+ cells from DDC liver were seeded on type-I collagencoated dishes in the presence of HGF, EGF and IL6. The morphology of the cells after 5 days of culture (A) and after several passages (B) is shown. (C) RT-PCR of EpCAM+ cells after 30 days of culture. Afp was strongly expressed in the cultured cells. (D) Immunostaining of the cultured cells with anti-CK19 and anti-ALB antibodies. DEVELOPMENT Characterization of the cell lines established from EpCAM+ cells As in freshly isolated EpCAM+ cells, CK19 and ALB were expressed in the established cell lines after 30 days of culture, as evidenced by RT-PCR and immunostaining (Fig. 6C,D). Afp, a marker of fetal liver progenitor cells, was expressed in the cultured cells (Fig. 6C), whereas it was not expressed in freshly isolated mouse oval cells, suggesting that more immature cells might be selected from EpCAM+ cells of NPCs in DDC liver. Alternatively, these proliferating cells might acquire a more hepatoblast-like character when cultured in vitro as previously reported (Schmelzer et al., 2007). In accordance with the latter idea, Epcam expression gradually decreased in culture (Fig. 6C). At the onset of mouse liver development, EpCAM is highly expressed in delta-like 1 homolog (DLK1)-positive hepatoblasts; however, its expression in hepatoblasts is dramatically reduced as liver development proceeds (our unpublished data). The reduction of EpCAM expression in cultured cells observed in this study is in line with the possibility that EpCAM+ cells might shift from an HSC-character to a more hepatoblast-like character. To investigate the bipotency of cultured hepatic stem-like cells derived from EpCAM+ cells (HSCEs) at the clonal level, we randomly picked several clones and finally established 12 independent cell lines (HSCE1-12). All the expanded clones expressed both hepatocytic (Alb) and cholangiocytic (Ck7, Ck19) genes (see Table S2 in the supplementary material). The expression of c-Met, which is known to be expressed in oval cells, was also detected in all clones. Whereas tyrosine aminotransferase (Tat), a marker of perinatal hepatocytes, was weakly expressed in some of the clones, glucose-6-phosphatase (G6Pase; G6pc), another marker of perinatal hepatocytes, and carbamoyl phosphate synthetase (Cps), an adult hepatocyte marker, were rarely expressed (2/12 and 0/12, respectively). Afp, an immature hepatocyte marker, was expressed in most of the clones (10/12). These results indicated that HSCEs maintain immature characteristics. Hepatic stem cells in adult liver RESEARCH ARTICLE 1957 Fig. 7. Clonally expanded HSCEs can differentiate into both hepatocytes and cholangiocytes. (A) Experimental design for the differentiation of hepatic stem-like cells derived from EpCAM+ cells (HSCEs) into hepatocytic cells. (B) RT-PCR of clone HSCE1 after hepatocytic differentiation. The addition of OSM, DMSO and EHS gel strongly induced the expression of hepatocytic genes and downregulated that of hepatoblastic and cholangiocytic genes. (C) PAS staining of HSCE1. The addition of OSM, DMSO and EHS gel strongly induced the accumulation of glycogen. (D) Morphological changes of HSCE1 after cholangiocytic differentiation. Tubules and branching morphology were clearly observed after 11 days of culture. (E) RT-PCR of HSCE1 after cholangiocytic differentiation. The expression of cholangiocytic marker genes was markedly upregulated in HSCE1. Differentiation of clonally expanded EpCAM+ cells into the cholangiocytic lineage in vitro To evaluate the potential for differentiation into cholangiocytes, we utilized a three-dimensional collagen gel culture that is effective for the formation of tubules (Nishikawa et al., 1996; Tanimizu et al., 2004a). A cell suspension in collagen type-I gel was plated onto a basal layer of a collagen type-I gel and then culture medium containing HGF, an inducer of tubulogenesis, was loaded on top. After 11 days of culture, the formation of tubules was observed, and after 18 days of culture branching structures were clearly evident (Fig. 7D). In addition, RT-PCR revealed that the expression of cholangiocytic marker genes was upregulated in the cultured cells (Fig. 7E), indicating that HSCEs differentiated into the cholangiocytic lineage. Thus, EpCAM was expressed on oval cells in DDC liver and the clonally expanded cells from sorted EpCAM+ cells exhibited characteristics of HSCs, i.e. unlimited proliferation and bi-directional differentiation. However, it remained unclear whether the emergence of such potential HSCs was an event restricted to liver injury and whether they were derived from oval cells. EpCAM+ cells in normal liver include potential HSCs Because EpCAM+ cells are present in normal mouse liver, we investigated whether potential HSCs are present among EpCAM+ cells of normal liver. Interestingly, the colony formation assay revealed that sorted EpCAM+ cells from normal liver formed both Fig. 8. Comparison between EpCAM+ cells isolated from normal and injured liver. (A) Colony formation assay of EpCAM+ cells from normal liver. Representative morphology of a small colony (top) and a large colony (bottom). Large colonies composed of more than 100 cells after 9 days of culture proliferate exponentially. (B) Unlimited cell proliferation of the established clones (#1-#4) in in vitro culture. (C) Comparison of cell surface markers between HSCEs from normal (blue line) and injured (red line) liver by FCM. Control IgG is in gray. The expression profiles of cell surface markers were similar in both HSCEs. (D) The number of EpCAM+ cells per normal (n=4) or injured (n=6) liver. The number was estimated from the percentage of EpCAM+ cells after immunomagnetic bead selection (see Fig. S2 in the supplementary material). There was a significant increase in EpCAM+ cells in DDC liver (*P<0.01). (E) Colony formation assay of EpCAM+ cells from normal and injured liver of mice fed DDC for 4 weeks. The data are derived from four independent experiments. Error bars, s.d. small and large colonies (Fig. 8A). These large colonies, which were composed of more than 100 cells, continued to proliferate, and these clones propagated continuously and exhibited bipotency similar to the HSCE clones derived from DDC liver (Fig. 8B and data not shown). In addition, HSCEs derived from normal and DDC liver exhibited a similar expression pattern of cell surface markers, strongly suggesting that they are closely related (Fig. 8C). By contrast, EpCAM– cells from normal or DDC liver did not give rise to any colonies, even when ten times more cells were plated than for EpCAM+ (data not shown). If oval cells themselves have characteristics of potential HSCs then oval cell activation by the DDC diet should increase the number of potential HSCs. To test this possibility, we compared the number of EpCAM+ cells and potential HSCs between normal and DDC liver (see Fig. S1 in the supplementary material). As shown in Fig. 8D, the number of DEVELOPMENT cells accumulating glycogen (Fig. 7C). These results strongly suggested that clonally expanded HSCEs differentiate into the hepatocytic lineage. EpCAM+ cells recovered from a DDC liver was approximately twice that from a normal liver. Interestingly, the numbers of large colonies corresponding to potential HSCs were very similar for normal and injured liver (Fig. 8E). In addition, adult potential HSCs are a very small population of EpCAM+ cells in normal and injured liver. Although we cannot exclude the possibility that potential HSCs might increase slightly (not more than 2-fold), it is unlikely that the activation of oval cells by liver injury significantly increases the number of potential HSCs. DISCUSSION Oval cells have been considered adult liver stem/progenitor cells (Fausto, 2004; Oertel and Shafritz, 2008). However, previous studies were mostly histochemical, or involved biochemical and molecular biological characterization using cell fractions prepared by density gradient techniques. FCM is a powerful means of characterizing a particular type of cell, as successfully demonstrated by the identification of a very rare population of hematopoietic stem cells using a combination of cell surface markers (Osawa et al., 1996). In fetal liver, we and others reported the isolation and characterization of hepatoblasts by FCM using several markers (Kubota and Reid, 2000; Suzuki et al., 2002; Tanimizu et al., 2003). Although definitive proof of stemness requires a clonal analysis of freshly isolated oval cells, a lack of specific markers has hampered the precise identification and prospective isolation of oval cells from NPCs by FCM. To address this issue, we first searched for cell surface molecules expressed on oval cells and showed that anti-EpCAM antibody is useful for isolating oval cells from the liver of DDC-fed mice by cell sorting. Clonal analyses provide strong evidence that the EpCAM+ cells from DDC liver contain adult potential HSCs that possess the capacity for unlimited proliferation and bi-directional differentiation. Hepatic stem/progenitor cells have been considered to contribute to the regeneration of damaged liver when the proliferation of hepatocytes is restricted, and cell lines with HSC characteristics have been established from chemically damaged and/or genetically dysfunctional liver (Braun et al., 1987; Dumble et al., 2002; Sugiyama et al., 1997; Suzuki et al., 2008; TirnitzParker et al., 2007; Yin et al., 1999). However, it remained unclear whether such cells are present in normal adult liver. Cell lines with HSC characteristics have been established from normal mouse (Fougere-Deschatrette et al., 2006), human (Herrera et al., 2006) and rat (Sahin et al., 2008) liver. However, these cell lines were derived from a hepatocyte-enriched cell population by centrifugation using a Percoll gradient or from unfractionated cells, leaving their origin ambiguous. Recently, Schmelzer et al. reported that EpCAM+ cells from postnatal human donors include HSCs (Schmelzer et al., 2007). In this report, we demonstrate that EpCAM+ cells isolated from normal or DDC mouse liver by FCM include potential HSCs. Interestingly, only a limited population of EpCAM+ cells formed large colonies and the number of potential HSCs was not significantly increased in DDC liver compared with normal liver. Thus, our results strongly suggest that most mouse oval cells do not possess self-renewal potential and are likely to represent transit-amplifying cells that differentiate into mature hepatic cells. Cholangiocytes proliferate under various pathological conditions. Cholangiocytes proliferate from pre-existing ducts in portal areas after PH or bile duct ligation in rats. Oval cell proliferation or ductular hyperplasia is induced by a number of chemicals, including 2-AAF, DDC (Preisegger et al., 1999) and by a choline-deficient ethionine-supplemented diet (Tian et al., 1997), and the proliferating Development 136 (11) cells form disorganized tubular structures that sprout into liver lobules. Selective damage of the periportal zone reduces such proliferation, supporting the notion that oval cells derive from the periportal region, in particular from the canals of Hering that connect the bile canaliculus and the biliary tree (Paku et al., 2001). However, the origin of oval cells is controversial and bone marrow stem cells were suggested to be a source (Petersen et al., 1999; Sell, 2001), although several reports refute this possibility (Menthena et al., 2004; Wang et al., 2003). Oval cells and cholangiocytes are not clearly distinguishable at the molecular level. In fact, oval cells and normal cholangiocytes are known to express many intracellular and membrane proteins in common, including EpCAM and CD133. However, it is also reported that some genes are predominantly expressed in rat or mouse oval cells. The expression of THY1, c-KIT and CD34 in oval cells has been controversial and these are also found in hematopoietic and mesenchymal cells (Dezso et al., 2007; Yovchev et al., 2008). By contrast, TROP2 is not expressed at all in normal liver and only in the oval cells of injured liver, indicating that TROP2 is a useful marker for oval cells. Intriguingly, after oval cell activation, most of the EpCAM+ cells express TROP2, and the original normal cholangiocytes, i.e. the EpCAM+ TROP2– cells, are hardly detected by FCM and immunostaining. These results raise the possibility that not only oval cells but also activated cholangiocytes might begin to express TROP2 after liver injury and partly contribute to oval cell proliferation in this mouse model (Fig. 9). Alternatively, oval cells that are activated to proliferate during DDC administration and that express both EpCAM and TROP2 might continue to express both of these markers at least transiently after they differentiate into mature cholangiocytes. Further investigation into the expression profile of TROP2 in cholangiocytes would provide a clue as to the origin of oval cells. Moreover, the function of EpCAM and TROP2 in oval cell activation is an interesting issue that remains to be addressed. Fig. 9. Model of the hepatic stem/progenitor cell system in vivo and in vitro. Potential HSCs exist in normal liver as EpCAM+ cells. They can proliferate unlimitedly and differentiate into both hepatocytes and cholangiocytes in vitro. Upon liver injury without oval cell activation, hepatocytes proliferate and contribute to liver regeneration. Upon liver injury with oval cell activation, EpCAM+ TROP2+ cells appear around portal veins to regenerate the liver. The oval cells might be partly derived from EpCAM+ cholangiocytes. Most oval cells lacked the potential to self-renew in the in vitro colony formation assay. DEVELOPMENT 1958 RESEARCH ARTICLE Oval cells are believed to be involved in the regeneration of liver following injury. In addition, they are also considered as a cellular precursor for hepatocellular carcinoma (HCC) (Knight et al., 2005). Most of the experimental procedures used to induce oval cell proliferation in the liver ultimately lead to tumorigenesis. However, the connection between oval cells and tumorigenesis remains unclear. EpCAM is expressed on many normal as well as neoplastic epithelial cells and tumor-initiating cells (Al-Hajj et al., 2003; Armstrong and Eck, 2003; Momburg et al., 1987). Most recently, Maetzel et al. reported that EpCAM is a potent signal transducer and that its cleaved intracellular domain utilizes components of the Wnt pathway to induce cell proliferation (Maetzel et al., 2009). Such signaling via EpCAM might be involved in oval cell proliferation. Conversely, Epcam is itself a Wnt/β-catenin signaling target gene in HCC cell lines (Yamashita et al., 2007). Consistently, the involvement of the Wnt/β-catenin pathway in the oval cell response has been reported (Apte et al., 2008; Hu et al., 2007; Yang et al., 2008). We recently reported that Wnt7a, Wnt7b and Wnt10a are upregulated in DDC liver and that the Wnt/β-catenin pathway is likely to be involved in oval cell activation in vivo (Itoh et al., 2009). Therefore, the correlation between tumorigenesis and the expression of EpCAM in oval cells is plausible. Because EpCAM is widely expressed in normal epithelial cells, including biliary epithelial cells, the expression and signaling of EpCAM should be tightly regulated at the steady state. However, the regulation of EpCAM signaling in normal and malignant stem-like cells remains unknown. It is also reported that TROP2 is expressed on cells of some normal tissues as well as on cancer cells (El Sewedy et al., 1998; Huang et al., 2005; Ohmachi et al., 2006). Our finding that TROP2, a member of the EpCAM family, is upregulated in oval cells raises the possibility that TROP2 might modulate and/or enhance the intracellular signaling of EpCAM to promote proliferation and migration into liver parenchyma. Alternatively, TROP2 itself might transduce intracellular signaling in a manner similar to EpCAM. In fact, ectopic expression of TROP2 in NIH3T3 cells is sufficient to promote both anchorage-independent growth and tumorigenesis (Wang et al., 2008). Most recently, Goldstein et al. reported that TROP2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics (Goldstein et al., 2008), suggesting an association between TROP2 and stem/progenitor cells. Although chronic liver injury induces the proliferation of oval cells in some rodent models and in human disease, the underlying relationship between oval cells and HSCs and their developmental mechanisms require further investigation. We showed the presence of potential HSCs in normal liver, as well as in injured liver, by isolating EpCAM+ cells and demonstrated the expression of TROP2 in oval cells. Further investigation into the interaction between EpCAM and TROP2 in oval cells will help us to understand the mechanisms of growth and differentiation in cancer-initiating cells as well as in normal stem/progenitor cells. Anti-A6 antibody was kindly provided by Dr Snorri Thorgeirsson and Dr Valentina Factor. This work was supported in part by a grant for Core Research for Evolutionary Science and Technology from the Japan Science and Technology Agency (JST) and a research grant from the Ministry of Education, Sports, Science and Technology (Japan). Supplementary material Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/11/1951/DC1 References Akhurst, B., Croager, E. J., Farley-Roche, C. A., Ong, J. K., Dumble, M. L., Knight, B. and Yeoh, G. C. (2001). A modified choline-deficient, ethionine- RESEARCH ARTICLE 1959 supplemented diet protocol effectively induces oval cells in mouse liver. Hepatology 34, 519-522. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. and Clarke, M. F. (2003). Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983-3988. Alison, M. R., Golding, M., Sarraf, C. E., Edwards, R. J. and Lalani, E. N. (1996). Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells. Gastroenterology 110, 1182-1190. Apte, U., Thompson, M. D., Cui, S., Liu, B., Cieply, B. and Monga, S. P. (2008). Wnt/beta-catenin signaling mediates oval cell response in rodents. Hepatology 47, 288-295. Armstrong, A. and Eck, S. L. (2003). EpCAM: A new therapeutic target for an old cancer antigen. Cancer Biol. Ther. 2, 320-326. Braun, L., Goyette, M., Yaswen, P., Thompson, N. L. and Fausto, N. (1987). Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats. Cancer Res. 47, 4116-4124. de Boer, C. J., van Krieken, J. H., Janssen-van Rhijn, C. M. and Litvinov, S. V. (1999). Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J. Pathol. 188, 201-206. Dezso, K., Jelnes, P., Laszlo, V., Baghy, K., Bodor, C., Paku, S., Tygstrup, N., Bisgaard, H. C. and Nagy, P. (2007). Thy-1 is expressed in hepatic myofibroblasts and not oval cells in stem cell-mediated liver regeneration. Am. J. Pathol. 171, 1529-1537. Dumble, M. L., Croager, E. J., Yeoh, G. C. and Quail, E. A. (2002). Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma. Carcinogenesis 23, 435-445. El Sewedy, T., Fornaro, M. and Alberti, S. (1998). Cloning of the murine TROP2 gene: conservation of a PIP2-binding sequence in the cytoplasmic domain of TROP-2. Int. J. Cancer 75, 324-330. Engelhardt, N. V., Factor, V. M., Medvinsky, A. L., Baranov, V. N., Lazareva, M. N. and Poltoranina, V. S. (1993). Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse. Differentiation 55, 19-26. Evarts, R. P., Nagy, P., Marsden, E. and Thorgeirsson, S. S. (1987). A precursorproduct relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8, 1737-1740. Farber, E. (1956). Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3⬘-methyl-4dimethylaminoazobenzene. Cancer Res. 16, 142-148. Fausto, N. (2004). Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39, 1477-1487. Fonia, O., Weizman, R., Coleman, R., Kaganovskaya, E. and Gavish, M. (1996). PK 11195 aggravates 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced hepatic porphyria in rats. Hepatology 24, 697-701. Fougere-Deschatrette, C., Imaizumi-Scherrer, T., Strick-Marchand, H., Morosan, S., Charneau, P., Kremsdorf, D., Faust, D. M. and Weiss, M. C. (2006). Plasticity of hepatic cell differentiation: bipotential adult mouse liver clonal cell lines competent to differentiate in vitro and in vivo. Stem Cells 24, 2098-2109. Gleiberman, A. S., Encinas, J. M., Mignone, J. L., Michurina, T., Rosenfeld, M. G. and Enikolopov, G. (2005). Expression of nestin-green fluorescent protein transgene marks oval cells in the adult liver. Dev. Dyn. 234, 413-421. Goldstein, A. S., Lawson, D. A., Cheng, D., Sun, W., Garraway, I. P. and Witte, O. N. (2008). Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proc. Natl. Acad. Sci. USA 105, 20882-20887. Hara, T., Nakano, Y., Tanaka, M., Tamura, K., Sekiguchi, T., Minehata, K., Copeland, N. G., Jenkins, N. A., Okabe, M., Kogo, H. et al. (1999). Identification of podocalyxin-like protein 1 as a novel cell surface marker for hemangioblasts in the murine aorta-gonad-mesonephros region. Immunity 11, 567-578. Herrera, M. B., Bruno, S., Buttiglieri, S., Tetta, C., Gatti, S., Deregibus, M. C., Bussolati, B. and Camussi, G. (2006). Isolation and characterization of a stem cell population from adult human liver. Stem Cells 24, 2840-2850. Hreha, G., Jefferson, D. M., Yu, C. H., Grubman, S. A., Alsabeh, R., Geller, S. A. and Vierling, J. M. (1999). Immortalized intrahepatic mouse biliary epithelial cells: immunologic characterization and immunogenicity. Hepatology 30, 358371. Hu, M., Kurobe, M., Jeong, Y. J., Fuerer, C., Ghole, S., Nusse, R. and Sylvester, K. G. (2007). Wnt/beta-catenin signaling in murine hepatic transit amplifying progenitor cells. Gastroenterology 133, 1579-1591. Huang, H., Groth, J., Sossey-Alaoui, K., Hawthorn, L., Beall, S. and Geradts, J. (2005). Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors. Clin. Cancer Res. 11, 4357-4364. Isom, H. C., Secott, T., Georgoff, I., Woodworth, C. and Mummaw, J. (1985). Maintenance of differentiated rat hepatocytes in primary culture. Proc. Natl. Acad. Sci. USA 82, 3252-3256. DEVELOPMENT Hepatic stem cells in adult liver Itoh, T., Kamiya, Y., Okabe, M., Tanaka, M. and Miyajima, A. (2009). Inducible expression of Wnt genes during adult hepatic stem/progenitor cell response. FEBS Lett. 583, 777-781. Jelnes, P., Santoni-Rugiu, E., Rasmussen, M., Friis, S. L., Nielsen, J. H., Tygstrup, N. and Bisgaard, H. C. (2007). Remarkable heterogeneity displayed by oval cells in rat and mouse models of stem cell-mediated liver regeneration. Hepatology 45, 1462-1470. Joplin, R., Strain, A. J. and Neuberger, J. M. (1990). Biliary epithelial cells from the liver of patients with primary biliary cirrhosis: isolation, characterization, and short-term culture. J. Pathol. 162, 255-260. Kamiya, A., Kinoshita, T., Ito, Y., Matsui, T., Morikawa, Y., Senba, E., Nakashima, K., Taga, T., Yoshida, K., Kishimoto, T. et al. (1999). Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J. 18, 2127-2136. Kamiya, A., Kojima, N., Kinoshita, T., Sakai, Y. and Miyaijma, A. (2002). Maturation of fetal hepatocytes in vitro by extracellular matrices and oncostatin M: induction of tryptophan oxygenase. Hepatology 35, 1351-1359. Knight, B., Matthews, V. B., Olynyk, J. K. and Yeoh, G. C. (2005). Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell. BioEssays 27, 1192-1202. Kojima, T. and Kitamura, T. (1999). A signal sequence trap based on a constitutively active cytokine receptor. Nat. Biotechnol. 17, 487-490. Kubota, H. and Reid, L. M. (2000). Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc. Natl. Acad. Sci. USA 97, 12132-12137. Laishes, B. A. and Rolfe, P. B. (1981). Search for endogenous liver colonyforming units in F344 rats given a two-thirds hepatectomy during short-term feeding of 2-acetylaminofluorene. Cancer Res. 41, 1731-1741. Lee, J. S., Heo, J., Libbrecht, L., Chu, I. S., Kaposi-Novak, P., Calvisi, D. F., Mikaelyan, A., Roberts, L. R., Demetris, A. J., Sun, Z. et al. (2006). A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat. Med. 12, 410-416. Lemaigre, F. P. (2003). Development of the biliary tract. Mech. Dev. 120, 81-87. Maetzel, D., Denzel, S., Mack, B., Canis, M., Went, P., Benk, M., Kieu, C., Papior, P., Baeuerle, P. A., Munz, M. et al. (2009). Nuclear signalling by tumour-associated antigen EpCAM. Nat. Cell Biol. 11, 162-171. Menthena, A., Deb, N., Oertel, M., Grozdanov, P. N., Sandhu, J., Shah, S., Guha, C., Shafritz, D. A. and Dabeva, M. D. (2004). Bone marrow progenitors are not the source of expanding oval cells in injured liver. Stem Cells 22, 1049-1061. Michalopoulos, G. K. and DeFrances, M. C. (1997). Liver regeneration. Science 276, 60-66. Momburg, F., Moldenhauer, G., Hammerling, G. J. and Moller, P. (1987). Immunohistochemical study of the expression of a Mr 34,000 human epithelium-specific surface glycoprotein in normal and malignant tissues. Cancer Res. 47, 2883-2891. Nishikawa, Y., Tokusashi, Y., Kadohama, T., Nishimori, H. and Ogawa, K. (1996). Hepatocytic cells form bile duct-like structures within a threedimensional collagen gel matrix. Exp. Cell Res. 223, 357-371. Notenboom, R. G., van den Bergh Weerman, M. A., Dingemans, K. P., Vermeulen, J. L., van den Eijnde, S., Reutelingsperger, C. P., Hut, H., Willemsen, R., Offerhaus, G. J. and Lamers, W. H. (2003). Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage. Hepatology 38, 683-691. Oertel, M. and Shafritz, D. A. (2008). Stem cells, cell transplantation and liver repopulation. Biochim. Biophys. Acta 1782, 61-74. Ohmachi, T., Tanaka, F., Mimori, K., Inoue, H., Yanaga, K. and Mori, M. (2006). Clinical significance of TROP2 expression in colorectal cancer. Clin. Cancer Res. 12, 3057-3063. Osawa, M., Hanada, K., Hamada, H. and Nakauchi, H. (1996). Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273, 242-245. Overturf, K., al-Dhalimy, M., Ou, C. N., Finegold, M. and Grompe, M. (1997). Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am. J. Pathol. 151, 1273-1280. Paku, S., Schnur, J., Nagy, P. and Thorgeirsson, S. S. (2001). Origin and structural evolution of the early proliferating oval cells in rat liver. Am. J. Pathol. 158, 1313-1323. Petersen, B. E., Goff, J. P., Greenberger, J. S. and Michalopoulos, G. K. (1998). Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27, 433-445. Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M., Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S. and Goff, J. P. (1999). Bone marrow as a potential source of hepatic oval cells. Science 284, 1168-1170. Preisegger, K. H., Factor, V. M., Fuchsbichler, A., Stumptner, C., Denk, H. and Thorgeirsson, S. S. (1999). Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease. Lab. Invest. 79, 103-109. Roskams, T. A., Theise, N. D., Balabaud, C., Bhagat, G., Bhathal, P. S., Bioulac-Sage, P., Brunt, E. M., Crawford, J. M., Crosby, H. A., Desmet, V. et Development 136 (11) al. (2004). Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology 39, 1739-1745. Rountree, C. B., Barsky, L., Ge, S., Zhu, J., Senadheera, S. and Crooks, G. M. (2007). A CD133-expressing murine liver oval cell population with bilineage potential. Stem Cells 25, 2419-2429. Sahin, M. B., Schwartz, R. E., Buckley, S. M., Heremans, Y., Chase, L., Hu, W. S. and Verfaillie, C. M. (2008). Isolation and characterization of a novel population of progenitor cells from unmanipulated rat liver. Liver Transpl. 14, 333-345. Sakai, Y., Jiang, J., Kojima, N., Kinoshita, T. and Miyajima, A. (2002). Enhanced in vitro maturation of fetal mouse liver cells with oncostatin M, nicotinamide, and dimethyl sulfoxide. Cell Transplant. 11, 435-441. Schmelzer, E., Zhang, L., Bruce, A., Wauthier, E., Ludlow, J., Yao, H. L., Moss, N., Melhem, A., McClelland, R., Turner, W. et al. (2007). Human hepatic stem cells from fetal and postnatal donors. J. Exp. Med. 204, 1973-1987. Seglen, P. O. (1976). Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29-83. Sell, S. (2001). Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology 33, 738-750. Sugiyama, K., Kato, N., Mizutani, T., Ikeda, M., Tanaka, T. and Shimotohno, K. (1997). Genetic analysis of the hepatitis C virus (HCV) genome from HCVinfected human T cells. J. Gen. Virol. 78, 329-336. Suzuki, A., Zheng, Y. W., Kaneko, S., Onodera, M., Fukao, K., Nakauchi, H. and Taniguchi, H. (2002). Clonal identification and characterization of selfrenewing pluripotent stem cells in the developing liver. J. Cell Biol. 156, 173-184. Suzuki, A., Sekiya, S., Onishi, M., Oshima, N., Kiyonari, H., Nakauchi, H. and Taniguchi, H. (2008). Flow cytometric isolation and clonal identification of selfrenewing bipotent hepatic progenitor cells in adult mouse liver. Hepatology 48, 1964-1978. Tanimizu, N., Nishikawa, M., Saito, H., Tsujimura, T. and Miyajima, A. (2003). Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J. Cell Sci. 116, 1775-1786. Tanimizu, N., Saito, H., Mostov, K. and Miyajima, A. (2004a). Long-term culture of hepatic progenitors derived from mouse Dlk+ hepatoblasts. J. Cell Sci. 117, 6425-6434. Tanimizu, N., Tsujimura, T., Takahide, K., Kodama, T., Nakamura, K. and Miyajima, A. (2004b). Expression of Dlk/Pref-1 defines a subpopulation in the oval cell compartment of rat liver. Gene Expr. Patterns 5, 209-218. Theise, N. D., Saxena, R., Portmann, B. C., Thung, S. N., Yee, H., Chiriboga, L., Kumar, A. and Crawford, J. M. (1999). The canals of Hering and hepatic stem cells in humans. Hepatology 30, 1425-1433. Tian, Y. W., Smith, P. G. and Yeoh, G. C. (1997). The oval-shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and immunohistochemical staining for albumin and pyruvate kinase isoenzyme expression. Histochem. Cell Biol. 107, 243-250. Tirnitz-Parker, J. E., Tonkin, J. N., Knight, B., Olynyk, J. K. and Yeoh, G. C. (2007). Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet. Int. J. Biochem. Cell Biol. 39, 2226-2239. Wang, J., Day, R., Dong, Y., Weintraub, S. J. and Michel, L. (2008). Identification of Trop-2 as an oncogene and an attractive therapeutic target in colon cancers. Mol. Cancer Ther. 7, 280-285. Wang, X., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M. and Grompe, M. (2003). The origin and liver repopulating capacity of murine oval cells. Proc. Natl. Acad. Sci. USA 100 (Suppl. 1), 11881-11888. Watanabe, N., Tanaka, M., Suzuki, K., Kumanogoh, A., Kikutani, H. and Miyajima, A. (2007). Tim2 is expressed in mouse fetal hepatocytes and regulates their differentiation. Hepatology 45, 1240-1249. Went, P. T., Lugli, A., Meier, S., Bundi, M., Mirlacher, M., Sauter, G. and Dirnhofer, S. (2004). Frequent EpCam protein expression in human carcinomas. Hum. Pathol. 35, 122-128. Yamashita, T., Budhu, A., Forgues, M. and Wang, X. W. (2007). Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res. 67, 10831-10839. Yang, W., Yan, H. X., Chen, L., Liu, Q., He, Y. Q., Yu, L. X., Zhang, S. H., Huang, D. D., Tang, L., Kong, X. N. et al. (2008). Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells. Cancer Res. 68, 4287-4295. Yin, L., Lynch, D. and Sell, S. (1999). Participation of different cell types in the restitutive response of the rat liver to periportal injury induced by allyl alcohol. J. Hepatol. 31, 497-507. Yovchev, M. I., Grozdanov, P. N., Joseph, B., Gupta, S. and Dabeva, M. D. (2007). Novel hepatic progenitor cell surface markers in the adult rat liver. Hepatology 45, 139-149. Yovchev, M. I., Grozdanov, P. N., Zhou, H., Racherla, H., Guha, C. and Dabeva, M. D. (2008). Identification of adult hepatic progenitor cells capable of repopulating injured rat liver. Hepatology 47, 636-647. Zaret, K. S. (2000). Liver specification and early morphogenesis. Mech. Dev. 92, 83-88. DEVELOPMENT 1960 RESEARCH ARTICLE
© Copyright 2020


text:
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פיתוח מיומנויות
Ashes of Creation hosts a wide array of progression paths that offer augments to a character's primary skills.[1][2]
- The progression system for augments is very similar to the class progression system.[3]
תוכן עניינים
Class augments
Primary skills (class abilities) are based on a player's archetype.[7] Players can personalize their primary skills with augmentation from a secondary archetype.[8][7][9]
The design behind augments is to not just change the flavor so that it reflects the secondary archetype, but it also fundamentally changes the core components of a skill.[10] – Steven Sharif
- Each secondary archetype offers four different schools of augmentation.[8][11] Each augment school affects a primary archetype's skills in different ways.[12]
- For example: A Mage offers Teleportation and elemental schools of augments. These augments will affect a Fighter's primary skills differently than a Cleric's.[8][12]
- Each augmentation has a level requirement and number of skill points required to activate it.[13]
- There's going to be a certain threshold at which you can no longer augment your active abilities based on the decisions you've augmented previous abilities, so you'll have to pick and choose which ones you want to apply the augments towards; and certain augments will have more expense required on the skill point side.[13] – Steven Sharif
- Augments to primary skills will fundamentally change the way the ability works: Adapting what the ability once did to incorporate the identity of the secondary archetype.[15]
- The progression system for augments is very similar to the class progression system.[3]
- Changing the augmentations on your skills will require you to go to a NPC in a Village node or higher.[16]
- Some spell colors and general FX change based on augments.[17]
- Active skills could look totally different after an augment gets applied.[18]
Players receive skill points as they level. These can be used to level up skills within their active, passive or combat skill trees.[19][20][21]
- It will not be possible to max all skills in a skill tree.[21]
- In terms of skill progression, players can choose to go "wide" and get a number of different abilities, or go "deep" into a few specific abilities.[22]
If from the eight archetypes whatever you choose as your secondary, you're going to receive a choice of augments that relate to some core ideal of that class. You know like a tank is about controlling the battlefield, is about surviving. The mage is about dealing damage and elements and ability in AoEs. The rogue is going to be about stealth and critical damage. So those augments are going to to play towards those identities.[23] – Steven Sharif
The idea behind the system is that you're kind of skirting the line through these augmentations of your role, right. We have the traditional holy trinity that's present in class designs for MMOs and it's often that those either are not deviated at all or completely deviated from entirely. The augment is to kind of offer a balance between that where you still maintain the semblance of that trinity system while offering the opportunity to customize your play experience towards one of the other angles in the triangle.[24] – Steven Sharif
Racial augments
Racial augments are based on a character's race and are available to each class.[25]
- These apply to abilities on a character's primary archetype.[26]
- They affect how the ability works and can also apply cosmetic effects.[27]
In keeping with our theme of Consequence, and unlike other games, race won't just be a matter of look, with relatively inconsequential abilities tacked on. Instead they integrate deeply into our class system, augmenting and changing the basic chassis provided by our eight core archetypes. Our goal here is to create a system where an elven fighter feels different, but serves the same role as a dwarven fighter.[28]
Religious augments
Strong religions provide unique augments that can be applied to a player's primary skills.[29][30]
- Religious augments are considered top-tier achievements within a religion.[29]
- These augments can be stacked on top of class augments. They will have a give-and-take system based on the choices a player makes.[29]
You're going to have advancement within the religion and within the church that you're a part of. And those advancements are going to be both reflective of the community's development of that religion out in the world: Building temples, building infrastructure for that, completing certain quests for it; and then you will benefit from the community's development of that specific religion via having certain augment abilities that you'll gain access to be able to apply on top of your secondary class augments to skills; and it's going to reflect what the nature of that religion is.[31] – Steven Sharif
Social organization augments
A player progresses through a social organization by accomplishing tasks or quests.[32] There are hierarchical paths pertaining to specific questlines for the organization's thematic.[32] These quests will either be cooperative with or adversarial against other nodes based on their mutual war status.[33]
- - For example, a Thieves' guild may have objectives and quests toward securing a particular item to enable players to advance within the organization.[32]
Social organizations are going to have questlines that players participate in which some will include sabotage, espionage, intrigue... While it's not necessarily player versus player in the combat sense, it is player versus player in pitting communities in those organizations kind of against each other in a competitive atmosphere, where only some things can be accomplished by certain communities; and not everybody can succeed at a particular task. So, I think that that's a unique way to involve meaningful conflict that doesn't necessarily have to relate to PvP, because obviously we have a lot of PvP systems in the game and and there are many ways for players to participate in player versus player combat; but we also want to make sure that from a progression standpoint, from a system standpoint there are going to be abilities of individuals to follow these questlines, these tasks that will pit organizations against each other, specifically from an organizational standpoint.[34] – Steven Sharif
- Social organizations unlock augments.
You're gonna see arguments being used a lot throughout a lot of our systems. When we're talking about the guild system, when we're talking about social organizations, when we're talking about religions. A lot of those things are going to affect how and what you have available to augment your skills with.[31] – Jeffrey Bard
Skill transfer
Players will not be able to transfer their skills to other players.[35]
See also
- ↑
- ↑ Official Livestream - May 4th @ 3 PM PST - Q&A
- ↑ 3.0 3.1 שידור חי, 2019-07-26 (1:09:22).
- ↑ שידור חי, 2020-03-28 (1:25:32).
- ↑ שידור חי, 2020-07-25 (58:38).
- ↑ וִידֵאוֹ, 2017-02-07 (0:02s).
- ↑ 7.0 7.1
- ↑ 8.0 8.1 8.2 רֵאָיוֹן, 2020-07-18 (1:05:04).
- ↑
- ↑ שידור חי, 2020-07-25 (1:47:55).
- ↑ שידור חי, 2018-02-09 (41:56).
- ↑ 12.0 12.1 שידור חי, 2019-12-17 (1:13:14).
- ↑ 13.0 13.1 רֵאָיוֹן, 2020-07-18 (1:07:06).
- ↑
- ↑ February 8, 2019 - Questions and Answers.
- ↑ שידור חי, 8 April 2018 (PM) (20:45).
- ↑
- ↑ רֵאָיוֹן, 2018-05-11 (53:15).
- ↑ רֵאָיוֹן, 2020-07-19 (53:59).
- ↑ רֵאָיוֹן, 2020-07-18 (1:07:51).
- ↑ 21.0 21.1 שידור חי, 2017-07-28 (19:05).
- ↑ שידור חי, 2017-11-16 (30:02).
- ↑ שידור חי, 2017-10-16 (1:00:44).
- ↑ רֵאָיוֹן, 2018-08-08 (22:27).
- ↑
- ↑ שידור חי, 2017-05-08 (43:30).
- ↑ שידור חי, 2019-07-26 (1:28:31).
- ↑ Ashes of Creation Kickstarter.
- ↑ 29.0 29.1 29.2 שידור חי, 2020-07-25 (57:02).
- ↑ שידור חי, 2017-05-08 (44:51).
- ↑ 31.0 31.1 שידור חי, 2017-06-01 (31:47).
- ↑ 32.0 32.1 32.2 שידור חי, 2017-05-17 (7:27).
- ↑ רֵאָיוֹן, 2020-07-19 (24:34).
- ↑ 34.0 34.1 34.2 34.3 Podcast, 2018-05-11 (18:52).
- ↑ שידור חי, 2017-11-17 (27:35).

subset: commoncrawl


text: search Search keywords City, state, zip code Find a job Please select an auto suggested location. Sort By Relevance Date within 25 miles Category Medical & Science Medical & Science 2 Medical Technician 2 Industrial & Manufacturing 1 State Florida Florida 2 City Thonotosassa Deerfield Beach 1 Fort Myers 1 Framingham 1 Ft Lauderdale 2 Holyoke 5 Jacksonville 4 Kissimmee 2 Lake Mary 1 Maimi 1 Melbourne 8 Miami 12 Naples 1 Opa Locka 1 Orlando 4 Quincy 9 Sarasota 1 Tampa 17 Temple Terrace 1 Thonotosassa 2 Waltham 1 West Melbourne 2 Contract Type Contract/Temporary Contract/Temporary 2 Category: customer-service State: florida City: fort-lauderdale Contract Type: Contract/Temporary Category: Medical & Science City: Thonotosassa Reset Searching for Thonotosassa Florida Medical & Science jobs? We currently have 2 jobs available. Create a job alert Related Searches For - Tap here to refine jobs Show more Show 10 20 30 Results Per page Medical Assistant - Thonotosassa, FL New Thonotosassa, Florida Contract/Temporary Specific Duties: 1.Room patients including vital signs 2. Clinical Intake 3.EKG 4.Lab Draws 5.Contacting patients by phone regarding normal lab work Must have qualifications/experience: 1.High school diploma or GED 2.Current nationally recognized MA... See job description Thonotosassa, FL - Certified Medical Assistant New Thonotosassa, Florida Contract/Temporary Certified Medical Assistant opportunities are now available in Thonotosassa, FL through Adecco Medical and Science. This is a long-term assignment, with the possibility of an extension or temp to perm opportunity. Specific Duties: 1.Room patients... See job description Clear your search criteria to see more results or browse the job categories we service. Send Us Your Resume and we will closely review your information. If we find an opportunity that matches your skills and career goals, we'll reach out to you to get to know you even better. Want new jobs? We'll email you. Name your job alert First name Last name Email How often would you like to see new jobs? Job Alert Duration Get the career advice you need when you need it. Sign up for our Career Advisor newsletter. Send me job alerts! By signing up. you are agreeing to the Terms of Use. Apply.JobAlertsTermsLabel Apply.TermsAndConditionsCheckBoxError Exit without saving × Awesome! Your job alert is saved. You'll see new jobs in your inbox soon. Good luck with your job search!


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Grey Bruce Ontario Provincial Police (OPP) is asking for the public assistance with a counterfeit money occurrence at a business at Highway 6 &10 west of Owen Sound.
On September 4, 2019 two individuals attended the business and made a small purchase. They used a $100 United States of America (US) bill to pay for the purchase and received the change, with exchange rate. It was discovered, later by store employees, that the US bill was counterfeit.
Attached is a photo of a person of interest. OPP are looking for the identity of the person in the photo.
Anyone with information that can assist Grey Bruce OPP can call 1-888-310-1122. Should you wish to remain anonymous, call Crime Stoppers at 1-800-222-8477 (TIPS) or submit a secure web-tip at www.cstip.ca, where you may be eligible to receive a cash reward of up to $2,000.
source: media release


text:
Ichalkaranji: 20.04.2016
Under the programme "One Nation One Mission" Terapanth Yuvak Parishad Ichalkaranji organized Mission Digital India Programme in the auspicious presence of Muni Suvrat Kumar as per instructed by ABTYP. In the programme Investment Consultant and Technology Specialist Sri Praveen Bhansali enlightened about online banking security, tips for online shopping, cyber safety etc. President Sri Sanjay Parasmal Vaid Mehta told about smart google search, useful smart apps and software, sites connected with open courseware, sites connected with blogging, online updating Aadhar Card (Base Card). Youngesters were present in a good number. Praveen Bhansali hosted the programme.
Mahila Mandal, Kishor Mandal, Kanyamandal, TYP at Mission Digital India Programme.


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Union minister Nitin Gadkari fainted at a public function at Solapur today. Gadkari, who was on a day-long tour of Solapur district today participated at the foundation day program of the Punyashlok Ahilyadevi Holkar Solapur University.
He fainted while the national anthem was being played at the end of the program. Gadkari stood up for the national anthem, but fainted and sat back to his seat even as the national anthem was just halfway through. He was immediately attended by doctors who declared him out of danger after preliminary examinations.
"Gadkari was supposed to attend two programs in the city. However, due to the health issues, he had to cancel all his programs after the program at Solapur University. He is now heading back to Nagpur," Maharashtra Minister for CoOperatives Subhash Deshmukh told The New Indian Express.
Gadkari arrived in Solapur at around 12 noon. After initial greetings at the airport, he headed directly to the University for the foundation day program. After attending the over 90-min program at the University, he fainted at the end of the program.
The other program, where he was to release a book written by cooperative minister Subhash Deshmukh, was then cancelled and Gadkari was rushed back to Nagpur after a rest of about an hour, said a close aide of Gadkari.


text:
Array Technologies' Solar Trackers Weather the Storm
Proven Solar Tracking Technology through Extreme Weather
ALBUQUERQUE, NM – 15 NOVEMBER 2018 – In 2018 the Eastern United States suffered two catastrophic hurricanes. Storms such as these can put solar installations at risk of catastrophic damage. When evaluating solar investments, a key consideration for solar farm projects is equipment reliability. Two of the most devastating storms in recent history, Hurricanes Florence and Michael, resulted in more than $25-billion in damages. Array Technologies today released a report surrounding the survivability of PV installations featuring the company's solar trackers that were caught in the eye of the storms.
"The devastation left behind by Florence and Michael was tragic, our thoughts and prayers go out to those who have been affected by these terrible disasters," said Jim Fusaro, chief executive officer at Array Technologies. "Planning and preparing for severe weather events is an absolute necessity, yet beyond this, there must be focused efforts on designing solutions that can withstand real-world conditions. At Array Technologies this is an area of focus for our engineers, ensuring our solar tracking solutions can survive severe weather and as was the case with hurricanes Michael and Florence, we did."
In mid-October, Georgia was struck by the most powerful hurricane it had seen since 1992, leaving a trail of devastation in its path. The category 4 Hurricane Michael caused more than $11-billion USD in damages, flattening crops, destroying roads, and damaging countless buildings. Hurricane Michael passed directly over Woodbine, GA, home to a solar installation utilizing Array's DuraTrack® HZ v3 single-axis solar trackers. The solar plant survived with zero damage.
In North Carolina, hundreds of megawatts of Array Technologies' trackers weathered a direct hit from Hurricane Florence. Standing up to maximum wind speeds of 130 mph followed by days of massive flooding, Array's DuraTrack® trackers came out unharmed, with systems fully operational. Senior leadership from the company attributes its trackers' ability to weather the storms to its patented wind-mitigation system which does not rely on complex communications systems, batteries, or power and flood clearance on all of its drive and control components.
"We are committed to engineering solar tracking solutions that can survive extreme weather conditions, providing our customers the reliability and returns needed to ensure solar generation remains the energy solution of the future," said Fusaro.
To read the report click here. To contribute to disaster relief in the affected areas, visit redcross.org.
About Array Technologies
Array Technologies, Inc. is the leading solar tracking solutions and services provider for utility-scale projects. With efficient installation and terrain flexibility coupled with high reliability, durability, and performance, Array delivers the best project returns in the industry. As the chosen tracker for more than 25 GW years of energy production, Array's products have been optimized through unparalleled experience garnered over nearly three decades. Array Technologies is headquartered in the United States with offices in Europe, Central and South America, the Middle East, and Australia. For more information, please visit https://arraytechinc.com/


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SWASHBUCKLING pirates will commandeer the Sprout Hub in Alkimos this Saturday for the launch of a business.
Pyjama Drama North West Perth will launch on September 23 with a pirate party for children aged up to seven.
Franchise manager Eleanor Mulder, of Tapping, said the event would include two interactive pirate shows where children can learn to talk like pirates, sail the seven seas, have fun with jungle animals and find buried treasure.
"There's no need to register – community members can turn up on the day for the family fun, which also includes a Pyjama Drama session for baby pirates up to two years, themed face-painting, parachute games," she said.
"Pyjama Drama offers drama and creative play classes for babies and children under seven years old.
"Igniting the imagination with drama games, sing-songs and role-playing, the classes develop a child's key social skills, such as concentration, co-operation, communication and confidence."
Mrs Mulder said children could dress up as pirates for the party, although their teachers did not use props or costumes.
"Children have the most amazing imaginations, they role-play easily and naturally, and pretending is actually the way in which children make sense of the world around them," she said.
The pirate party will run from 11am to 1pm at the Sprout Hub on Grasswren Way, Alkimos.
Pyjama Drama North West Perth can run classes at early learning centres, schools and parties between Carramar and Two Rocks.
MORE: VIDEO: Drone footage of whale and calf off Perth's coast
MORE: Curtin University law students offer services to small businesses pro bono
MORE: New signs designed to create smooth sailing for surfers


text:
HONG KONG-China Tire Holdings Ltd., the rapidly expanding independent Chinese tire manufacturing group, has acquired majority control of private brand distributor Orion Tire Corp., which will now become China Tire's spearhead in marketing its tires outside of China. Orion Tire founder Nicholas G. Clarke will continue as president, CEO and minority owner of the San Clemente, Calif.-based firm, which also owns Apollo Tire, a 17-store retail dealership in the Los Angeles area.
Orion Tire intends to market Orion-brand tires and other China Tire brands through distributors.


text:
Skylanders Giants: Chill (Xbox 360)
missing cover art
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DescriptionChill is a Water Element character figure for the game Skylanders Giants. Once a protector of the Snow Queen, she resigned and joined the Skylanders after her queen was kidnapped during Cyclops attacks. She has been on the lookout for her since. Once placed on the Portal of Power, Chill will become the player character. She uses her twin ice javelin and can control ice to fight.
The Chill figure is also available in a three-characters pack together with Ignitor and Zook.
There are no Xbox 360 user screenshots for this game.
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- Skylanders Giants: Chill (official website)
Fred VT (25924) added Skylanders Giants: Chill (Xbox 360) on Jul 30, 2014
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text:
In some parts of the world, the island-studded fiord called Howe Sound would have been locked up as a national park long ago, given its astounding natural beauty on the edge of a metropolis of more than 2.5-million people.
It's a special place where steep-sided mountains plunge almost 300 metres into glacier-fed waters that are home to a wide range of marine life, including salmon, herring, whales, dolphins, porpoises, and fragile glass-sponge reefs.
tap here to see other videos from our team.
But full protection is not what happened to Howe Sound.
Industry indelibly made its mark on the shoreline in 1904, with the opening of the Britannia mine, toasted as the "largest copper mine in the British Commonwealth." The mine closed in 1974. But it lives on today as a national historic site and tourist attraction clinging to a hillside and as a continuing source of so much pollution that a treatment facility had to be built in 2006, with a budget of $3 million a year to remove an average of 226,000 kilograms of heavy metal contaminants each year.
Over the decades, industry continued to come and go in the sound, including the Western Forest Products Woodfibre pulp mill, closed in 2006, on the same site where B.C. Sulphite Fibre Company began operations in 1912.
The place remains a contamination nightmare.
The list of nasties includes lead paint, asbestos, an old landfill that needs to be capped and closed, and some 3,000 creosote pilings that must be removed without stirring up old contaminants lurking in sediments on the ocean bottom.
"A lot of crap," confirms Byng Giraud, who is managing construction of the proposed $1.6-billion Woodfibre liquefied natural gas facility on the old pulp grounds that would ship out 2.1 million tonnes of product per year. "You need deep pockets to take over one of these old abandoned sites. We've inherited all those liabilities."
The other major industrial development gearing up along Howe Sound is the Burnco Rock Products Ltd. gravel mine at McNab Creek, a salmon spawning stream. The company promises to enhance the site to create better habitat for salmon.
Residents fiercely protective of 44-kilometre-long Howe Sound are tired of fighting one industrial project after another, but they aren't giving up.
"We go from defeat to defeat with undiminished enthusiasm," said Eoin Finn, co-founder of the citizens' group My Sea to Sky.
The closure of Woodfibre pulp mill and opening of the Britannia treatment facility, along with herring habitat improvements by the community, are considered largely responsible for an ecological re-awakening of the sound at all levels of the food chain. Salmon have even found their way back to Britannia Creek to spawn.
The fear is that these environmental gains are now threatened by industry's hand.
Finn is a retired partner with KPMG who lives in Kitsilano but has a property on Boyer Island, between Lions Bay and Horseshoe Bay. He would like to see Howe Sound declared a no-go zone for industry, and reserve the fiord for nature and the recreational enjoyment of people. The sound could be managed collectively by local municipal governments, with senior governments compensating industry for lost opportunities, he suggested.
"It's the re-industrialization of Howe Sound," he complained. "It's not just NIMBY. This is a resource that all of Vancouver enjoys and the people who live up and down the sound use. They have a right not to have it re-industrialized."More motorists, including tourists taking side trips from Vancouver to Squamish and Whistler, are using the corridor all the time. According to the Ministry of Transportation and Infrastructure, traffic on the Sea to Sky Highway has increased 24 per cent — a daily average of 19,000 vehicle trips through Lions Bay and Squamish and 10,000 through Whistler — since 2009 when the province completed $600 million in highway improvements for the 2010 Winter Olympics.
Industry has different thoughts.
During a Postmedia News tour of the Woodfibre site, Giraud argued that industrial operations have a long history in Howe Sound and that liquefied natural gas has a good environmental track record internationally. This particular project will operate on hydroelectricity from B.C. Hydro and on is an isolated stretch of the sound seven kilometres from downtown Squamish.
Unlike its pulp mill predecessor, the LNG plant also won't be pumping pollutants into the sound.
Giraud notes that the presence of industrial activities in Burrard Inlet has not diminished Vancouver's lure as a global tourist destination, evidence that industry and environment can work together. "I was born and raised in this province. There are lots of special and important places, but I don't think industry and tourism and special places are incompatible."
The project is predicted to employ about 100 workers during operation at salaries of about $100,000 a year. Some prospective employees already showing interest in a 1,000-unit housing development proposed for Britannia, right across the sound from the mine. Residential development is also being proposed for Furry Creek, a 10-minute drive south on Highway 99.
The Woodfibre LNG project is headed by Singapore billionaire Sukanto Tanoto of Royal Golden Eagle, a man with a checkered environmental record in Asia, including pulp operations accused by Greenpeace of destroying Indonesian rainforests.
"This is not the sort of character you'd invite to be your neighbour," Finn said. "We certainly don't in a place like Howe Sound."
Giraud responds that this is Canada, and Woodfibre LNG commits to meet or beat environmental requirements for its operation.
LNG accidents do happen. In 2017, a contractor was injured during a fire at a refrigerant line at a FortisBC LNG facility on Tilbury Island in Delta. In March 2014, six workers were injured in an explosion at the Williams Northwest Pipeline facility in Plymouth, Wash. Police said it was a miracle no one died as 100-plus-kilogram pieces of steel were tossed close to 100 metres.
Critics fear that a fiery explosion at Woodfibre LNG might spark a major forest fire, with a potentially worse disaster at sea. "The consequences would be massive," Finn said. "You'd toast Howe Sound."Woodfibre LNG hopes to break ground next year on a four-year construction project. KBR, an engineering and construction firm based in Houston, Texas, is expected to provide a detailed cost estimate of the project soon. A rebound in LNG prices recently makes Woodfibre more optimistic about its operation, whichwould ship out three or four laden LNG tankers a month, with tug escorts to the open ocean, to Japan and China.
The District of Squamish council has not voted on Woodfibre LNG, but a survey of councillors reveals a split on the issue."We do not need the LNG terminal and more ships coming up the inlet in our area," said councillor Ted Prior. "However, the jobs are needed in the Interior and Alberta, the gas is needed overseas. So I support." Councillor Karen Elliott counters: "I did not support Woodfibre LNG during the environmental assessment process and still believe this is the wrong industry for Howe Sound." Mayor Patricia Heintzman: "I do not think it is in an appropriate location. If these are developed they should be offshore, not at the end of a fiord and through a busy shipping route or in an area that is rebounding after a 100+ years of industrialization." Shipping in Howe Sound is not without risks. A barge sank in Howe Sound near Port Mellon in early 2018, and a fuel tank on board leaked up to 200 litres of diesel fuel into the sound.
For a perspective on vessel traffic in Howe Sound: Squamish Terminals, which sustained major damage in a waterfront fire in 2015, receives about five cargo ships a month, mostly carrying less incendiary products such as wood pulp and steel products.
Additional shipping, however, affects whales buy raising the potential for ship strikes and by increasing underwater noise levels.
At the Burnco site, south of Woodfibre, a family-owned Calgary company plans to start dredging McNab Creek next year. The site is on about 70 hectares of private land and would operate for 16 years at a maximum production of 1.6 million tonnes a year. The project will include a barge-loading site to move gravel to company operations in Langley and Burnaby.
Earlier this year, B.C.'s Environmental Assessment Office supported the project despite "direct impacts from the permanent loss of fish and wildlife habitat, and some uncertainties about the time it may take for migration and offsetting to become effective."
Critics say it's just another example of a flawed environmental process, one that inevitably favours industry.
The 16-megawatt Box Canyon Creek run-of-river hydroelectric project — about 10 kilometres from Port Mellon pulp mill near Gibsons, the last remaining such operation in the sound — went ahead without a full environmental assessment because it was not large enough to require one. The Elemental Energy Inc. project, partnered with Squamish First Nation, launched in 2016.
The Burnco fish mitigation plan calls for a net gain of more than 700 square metres of in-stream habitat and 21,209 square metres of riparian habitat to the south of the mine site.
While supporting the project, the Environmental Assessment Office noted: "Socio-economic impacts would be most noticeable in areas adjacent to the mine, where a number of effects may occur concurrently, such as changes to air quality, noise levels, visual quality and real estate values."
Critics sympathize with the owners of about 17 nearby recreational homes who came here for the peace and beauty. "It's angered a lot of people," Finn said. "Given how rich the B.C. coastline is in gravel deposits, why do they have to dig up a salmon bearing stream?"
Derek Holmes, B.C. land and resource manager for Burnco, said McNab Creek has several attractive attributes, including proximity to markets. "There are relatively few neighbours, it looks like it has the quality of sand and gravel deposits required for concrete, asphalt and general construction, and it seems large enough to be economically viable.
"It's easy to say no to everything, and gravel seems to be one of those hot topics, but I don't think people realize how much they use gravel, and how ubiquitous it is in our society. I'm curious to know how people … think you can do construction without it. Nobody wants it close to where they live or recreate. It's a tough balance."
Finn doesn't toss a wet blanket on all industrial operations. He believes logging practices have come a long way, employing smaller and more creative clearcuts that help to protect viewscapes. "It's a renewable resource and it's improved greatly," he said, adding that "logging is an industry that's been part of the B.C. coast for a long time."
Logging operations can also hurt the marine environment.
Environment Canada guidelines on log storage and handling warn such operations can cause local environmental damage if wood waste, such as bark and chips, get into the water. They can smother water plants, benthic invertebrates and fish eggs and also reduce the living space for juvenile fish.
Work is underway to document important marine sites that could be damaged by development.
Willem Van Riet, a spatial analyst for the David Suzuki Foundation, is working with the Vancouver Aquarium's Coastal Ocean Research Institute on a conservation assessment, a reference guide to the most important and vulnerable sites in the sound.
Started in June 2017, the assessment is scheduled for completion this September. "We want to identify ecologically unique areas, then lobby for their protection" Riet said. The results will be available online.
Ecological features include plant communities (kelp, eel grass and seaweed), important areas for marine life (fish, birds, mammals, invertebrates and reptiles), the physical environment (bathymetry, seabed classification, tides and currents), and marine use and activities (traditional uses and value, recreation, leases and harvesting).
Closer to Squamish, industrial impacts assume an unlikely face.The Squamish Spit, a long dike or "training dike" created for a coal port that never happened in the 1970s, is a popular area for wind sports. Reconfiguration of the dike, including a bridge, is under discussion to improve the area for juvenile salmon between the Squamish River and Squamish estuary, with a new access point for wind sports.
One might think there is no common ground to be found between conservationists and industry — but that's not entirely true.
Ruth Simons, a former Lions Bay councillor who heads the Future of Howe Sound Society, says it's time for a new way of thinking, a holistic approach that considers cumulative impacts — a big-picture view, starting now and into the future.
One option is to officially designate Howe Sound a UNESCO biosphere reserve, similar to Clayoquot Sound and Mount Arrowsmith, both on Vancouver Island. Planning has been going on for two years, with the aim of submitting the official nomination in 2018.
UNESCO says that a biosphere reserve, in part, "promotes solutions reconciling the conservation of biodiversity with its sustainable use" and includes "conflict prevention and management of biodiversity."
The label cannot block development — that isn't Simons' goal — but it would set a tone for sustainability, letting industry and anyone who seeks to develop know that they are entering a special area that requires extra attention.
"It would support more research and education," adds Simons, who regularly patrols the sound in her eight-metre power boat. "And anyone who came here would have an indication of what to expect."
Giraud says he could live with the designation, noting that sustainable logging continues in Clayoquot Sound. "A biosphere in not a park," he said. "We feel we could be compatible."B.C.'s Ministry of Forests, Lands, Natural Resource Operations and Rural Development is completing assessment reports on the cumulative effects of human activities on different natural resources around Howe Sound: aquatic ecosystems, visual-quality old-growth forests, forest biodiversity, grizzly bears, Roosevelt elk, and marbled murrelets. The reports are due for release this fall, and will likely set off a new debate on the future of Howe Sound. email@example.com


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Ohio moves toward Down syndrome abortion ban
The Ohio Senate passed a bill on a 20-12 vote to ban abortions solely based on a diagnosis of Down syndrome just weeks after the Ohio House passed a similar bill by a wide margin.
Ohio Right to Life has pushed for a ban on abortions related to Down syndrome for years, and the group's president, Mike Gonidakis, said last week that the latest developments were encouraging: "All Ohioans, regardless of their gender, skin color, or disability, deserve the right to live out their God-given potential and purpose."
Critics of the bill said it interferes with women's relationships with their doctors and pointed out it protects babies with Down syndrome but not other disabilities.
Ohio Gov. John Kasich has a record of approving some pro-life laws, but not all. Last year, he signed a 20-week abortion ban but nixed a ban on abortions after a baby has a detectable heartbeat. —S.G.