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Characterization of Proliferation and Tumorigenic Potential of Hepatic Oval Cells
- 1. Department of Surgery, University of Tsukuba
- 2. Viral hepatitis and liver cancer;M Levrero ;Nature review , 2006 Liver cancer as a result of chronic liver injury Department of Surgery, University of Tsukuba
- 4. Study overview Department of Surgery, University of Tsukuba Snapshot : Oval cell is considered the stem cell of liver . This study tries to investigate the proliferation characteristics, and tumorgenesis of oval cells . Male Sprague dawley rat was feed with choline deficient diet CDE(to induce oval cell )>>>hepatic oval cell isolation >>2 years culture :100 passage >> Flow cytometry –immunoflurence ( Ov6 , BD-2, BD-2, Dlk antibodies /markers)>>microscopic morphology study>proliferation ability > colony formation ability study >>> Xenograft into immunodeficient mouse to observe tumorgenesis . HepG2 cell line was used as control in this study .
- 5. Male Sprague dawley rat was feed with choline deficient diet CDE(to induce oval cell )-6 weeks hepatic oval cell isolation 2 years culture :100 passages study Xenograft into immunodeficient mouse to observe tumorgenesis . Department of Surgery, University of Tsukuba Research flow
- 6. Microscopic study Department of Surgery, University of Tsukuba
- 7. Markers • Hepatic progentior markers : OV-6, Bile duct (BD-1) , Bile duct 2(BD-2) , DLK(Delta like protein). • Colangiocyte maker :CK-19 • Hepatocyte marker : AFP , Albumin . • Sodium butyrate was given to induce hepatocytic differentiation .
- 8. Flow cytometry Department of Surgery, University of Tsukuba
- 9. Department of Surgery, University of Tsukuba
- 10. Department of Surgery, University of Tsukuba Oval cells treated with Sodium Butyrate
- 11. Department of Surgery, University of Tsukuba
- 12. Department of Surgery, University of Tsukuba Hepatic oval cell proliferation capability and chromosomal stability
- 13. Department of Surgery, University of Tsukuba Colony formation assessment: anchorage growth assessment
- 14. Department of Surgery, University of Tsukuba In vivo :xenograft to immunodeficient mouse
- 15. Department of Surgery, University of Tsukuba In summary, serial passages in vitro did not change the immunophenotype,proliferation capacity, or differentiation potential of hepatic oval cells and did not cause spontaneous maltransformation of these cells. Therefore, hepatic oval cells without immortalized manipulation not only provide an expandable cell source for future cell-therapy utilizations but also may serve as a cell model for understanding the mechanism of carcinogenesis This study also suggests that hepatic oval cells did not experience spontaneous maltransformations into cancer- initiating cells, suggesting the safety of utilizing hepatic stem/progenitor cells for exploitation of stem cell technology. Summary
Editor's Notes
- This diagram shows the pathogenesis of liver cancer : Chronic liver injury such as hepatitis B and C infection and alcohol can result liver hepatitis and cirrhosis and eventually cancer , this is a multistep progressive process lead to genetic alteration resulting dysplastic nodeules (preneoplastic nodules ) which later develop into cancer. Pathogenesis of human hepatocellular carcinoma (HCC). Chronic hepatitis B and C and associated liver cirrhosis represent major risk factors for HCC development, being implicated in more than 70% of HCC cases worldwide. Additional etiological factors, which often represent co-factors of an underlying HBV- or HCV-related chronic liver disease, include toxins and drugs (e.g., alcohol, aflatoxins, microcystin, anabolic steroids), metabolic liver diseases (e.g., hereditary hemochromatosis, alpha1-antitrypsin deficiency), steatosis, non-alcoholic fatty liver diseases and diabetes. Hepatocarcinogenesis is a multistep process that may last for decades and involves the progressive accumulation of different genetic alterations ultimately leading to malignant transformation. Regardless of the etiological agent, malignant transformation of hepatocytes is believed to occur through a pathway of increased liver cell turnover, induced by chronic liver injury and regeneration, in a context of inflammation and oxidative DNA damage. Dysplastic nodules and macroregenerative nodules are considered as pre-neoplastic lesions. The detailed analysis of HCC development in experimental animals and the comparison of the results with HCC in humans has identified a variety of genomic and molecular alterations in fully developed HCC and to a lesser extent in morphologically defined pre-neoplastic precursor lesions. At least four pathways that regulate either cell proliferation or cell death (i.e., the phospho-retinoblastoma (pRb), p53, transforming growth factor-beta (TGF-beta) and beta-catenin pathways) are affected in HCCs.
- Journal of Gastroenterology and Hepatology (2003) , REVIEW Oval cell-mediated liver regeneration: Role of cytokines and growth factors The 2-AFF induced the proliferation of a periportal population of oval like cells ( Negative for expression of classical oval cell markers ) .However , the expression of these markers (AFP) started after two days ( Hepato-lineage ) affliated subpopulation .
- Background & Aims: Although expandable hepatic progenitors provide renewable cell sources for treatment of hepatic disorders, long-term cultivation of hepatic progenitors may affect proliferation and differentiation abilities, and even initiate the formation of malignant cancer stem cells. This study aims to determine characteristics of primary cultured hepatic oval cells after prolonged cultivation in vitro.
- This will be the steps this experiment.
- 1. The typical epithelial morphology of hepatic oval cells after serial passages. (A) The morphology of hepatic oval cells under low phase-contrast microscope
- Fig. 2. Flow cytometry analysis demonstrating that hepatic oval cells expressed hepatic progenitor cell markers. The positive rate of the common marker for hepatic oval cells, OV-6, at primary culture, 10th, 50th, and 100th passage, was 87.2%, 77.7%, 81.1%, and 76.7%, respectively. The expression of BD-1, BD-2, and Dlk, was over 85%, 90%, and 98%, respectively. As a negative control, primary cultured hepatocytes did not express these hepatic progenitor cell markers.
- Fig. 3. Hepatic oval cells sustained expression of AFP, ALB, and CK19. (A) Immunofluorescence results showed that hepatic oval cells at the 0–2nd, 10th, 50th, and 100th passage expressed hepatocytic markers AFP and ALB. (B) Realtime RT-PCR results showed that hepatic oval cells had a sustained expression of AFP, ALB, and CK19. (C) Western blot results demonstrated that in hepatic oval cells the expression of AFP, ALB, and CK19 persisted.
- Fig. 4. Hepatocyte-specific differentiation capacity of hepatic oval cells after serial passages. (A) High power phase contrast images of hepatic oval cells exposed to sodium butyrate (0.75 mM) for 3 days, and stained for Giemsa or PAS; hepatic oval cells at different passages increase in cell size, become binuclear and/or polyploid and accumulate intracellular polysaccharides. Periodic acid–Schiff (PAS) is a staining method used to detect polysaccharides such as glycogen, and mucosubstances such as glycoproteins, glycolipids and mucins in tissues. (B) Western blot showing that the expression of ALB is upregulated, CK19 is slightly downregulated, while CK18 is significantly decreased after sodium butyrate (0.75 mM) inducement from different passages of oval cells. (C and D) Quantification of the albumin secretion and ureagenesis showing that albumin in the supernatant is increased and transformed urea is also increased at different passages of oval cells after sodium butyrate (0.75 mM) treatment.
- B) Western blot showing that the expression of ALB is upregulated, CK19 is slightly downregulated, while CK18 is significantly decreased after sodium butyrate (0.75 mM) inducement from different passages of oval cells. (C and D) Quantification of the albumin secretion and ureagenesis showing that albumin in the supernatant is increased and transformed urea is also increased at different passages of oval cells after sodium butyrate (0.75 mM) treatment.
- Fig. 5. Hepatic oval cells survive after serial passages without loss of their proliferation ability and maintain diploid cells with features of chromosomal stability. (A) Growth curves for the cell count of hepatic oval cells revealed that their doubling time at the 0–2nd, 10th, 50th, and 100th passage was 25.8, 25.4, 22.3, and 23.1 h, respectively. (B) Representative flow cytometry analysis of DNA content and cell cycle showing that hepatic oval cells sustain their proliferation potential and maintain the typical PI-staining pattern for diploid cells.
- Fig. 6. Colony formation in vitro and tumorigenesis in vivo. (A) HepG2 cells and hepatic oval cells at different passages plated in soft agar for 3 weeks. Unlike the positive control, tumorigenic HepG2 cells, hepatic oval cells at different passages fail to proliferate in soft agar. (B and C) Quantification by ELISPOT of the rate of formation of colonies (B), and the % of the well area covered (C) (*p <0.05) for hepatic oval cells, there is no significant difference in colony number and covered area at the 0–2nd, 10th, 50th, and 100th passage. (D) In vivo tumorigenesis by injection of different passages of oval cells subcutaneously in the left flank of the immunodeficient mouse (five mice per group); the positive control cells, HepG2, induce tumors after 2 and 4 weeks in the immunodeficient mice, while the oval cells cause no tumors.
- Fig. 6. Colony formation in vitro and tumorigenesis in vivo. (A) HepG2 cells and hepatic oval cells at different passages plated in soft agar for 3 weeks. Unlike the positive control, tumorigenic HepG2 cells, hepatic oval cells at different passages fail to proliferate in soft agar. (B and C) Quantification by ELISPOT of the rate of formation of colonies (B), and the % of the well area covered (C) (*p <0.05) for hepatic oval cells, there is no significant difference in colony number and covered area at the 0–2nd, 10th, 50th, and 100th passage. (D) In vivo tumorigenesis by injection of different passages of oval cells subcutaneously in the left flank of the immunodeficient mouse (five mice per group); the positive control cells, HepG2, induce tumors after 2 and 4 weeks in the immunodeficient mice, while the oval cells cause no tumors.