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Culture Conditions Promoting Hepatocyte Proliferation and Cell Cycle Synchronization

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Protocols in In Vitro Hepatocyte Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1250))

Abstract

The liver overcomes damages induced by harmful substances or viral infections and allows the use of extended resection in human therapy through its remarkable ability to regenerate. The regeneration process relies on the massive proliferation of differentiated hepatocytes that exit quiescence and undergo a limited number of cell cycles to restore the hepatic mass. Many discoveries on the regulation of hepatocyte proliferation have benefited from the use of in vitro models of cultures of primary hepatocytes as well as hepatoma cells as opposed to data obtained from in vivo models of liver regeneration, such as following partial hepatectomy in rodents. In this chapter, the most pertinent in vitro models used to promote the proliferation of hepatocytes and technical procedures to synchronize their progression throughout the cell cycle are presented with the goal to investigate the regulation of the hepatocyte cell cycle and the molecular pathways regulating liver regeneration.

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References

  1. Fausto N (2004) Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39:1477–1487

    Article  PubMed  Google Scholar 

  2. Michalopoulos GK, DeFrances M (2005) Liver regeneration. Adv Biochem Eng Biotechnol 93:101–134

    CAS  PubMed  Google Scholar 

  3. Avril A, Pichard V, Bralet MP et al (2004) Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury. J Hepatol 41:737–743

    Article  CAS  PubMed  Google Scholar 

  4. Higgins GM, Anderson RM (1931) Experimental pathology of liver: restoration of the liver of the white rat following partial surgical removal. Arch Pathol 12:186–202

    Google Scholar 

  5. Hsu JC, Bravo R, Taub R (1992) Interactions among LRF-1, JunB, c-Jun, and c-Fos define a regulatory program in the G1 phase of liver regeneration. Mol Cell Biol 12:4654–4665

    CAS  PubMed Central  PubMed  Google Scholar 

  6. Grisham JW (1962) A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver: autoradiography with thymidine-H3. Cancer Res 22:842–849

    CAS  PubMed  Google Scholar 

  7. Fabrikant JI (1968) The kinetics of cellular proliferation in regenerating liver. J Cell Biol 36:551–565

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Albrecht JH, Hoffman JS, Kren BT et al (1993) Cyclin and cyclin-dependent kinase 1 mRNA expression in models of regenerating liver and human liver diseases. Am J Physiol 265:G857–G864

    CAS  PubMed  Google Scholar 

  9. Fausto N, Campbell JS, Riehle KJ (2006) Liver regeneration. Hepatology 43:S45–S53

    Article  CAS  PubMed  Google Scholar 

  10. Cressman DE, Greenbaum LE, DeAngelis RA et al (1996) Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274:1379–1383

    Article  CAS  PubMed  Google Scholar 

  11. Webber EM, Bruix J, Pierce RH et al (1998) Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 28:1226–1234

    Article  CAS  PubMed  Google Scholar 

  12. Seglen PO (1975) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83

    Article  Google Scholar 

  13. Guguen-Guillouzo C, Guillouzo A (1986) Isolation of rat hepatocytes. In: Guillouzo A, Guillouzo C (eds) Isolated and cultured hepatocytes. INSERM, Paris, pp 1–12

    Google Scholar 

  14. Vanhaecke T, Rogiers V (2006) Hepatocyte cultures in drug metabolism and toxicological research and testing. Methods Mol Biol 320:209–227

    CAS  PubMed  Google Scholar 

  15. Etienne PL, Baffet G, Desvergne B et al (1988) Transient expression of c-fos and constant expression of c-myc in freshly isolated and cultured normal adult rat hepatocytes. Oncogene Res 3:255–262

    CAS  PubMed  Google Scholar 

  16. Loyer P, Cariou S, Glaise D et al (1996) Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro: evidence of a mitogen restriction point in mid-late G1. J Biol Chem 271:11484–11492

    Article  CAS  PubMed  Google Scholar 

  17. McGowan JA, Bucher NL (1983) Pyruvate promotion of DNA synthesis in serum-free primary cultures of adult rat hepatocytes. In Vitro 19:159–166

    Article  CAS  PubMed  Google Scholar 

  18. Sawada N (1989) Hepatocytes from old rats retain responsiveness of c-myc expression to EGF in primary culture but do not enter S phase. Exp Cell Res 181:584–588

    Article  CAS  PubMed  Google Scholar 

  19. Nakamura T, Nishizawa T, Hagiya M et al (1989) Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440–443

    Article  CAS  PubMed  Google Scholar 

  20. Garnier D, Loyer P, Ribault C et al (2009) Cyclin-dependent kinase 1 plays a critical role in DNA replication control during rat liver regeneration. Hepatology 50:1946–1956

    Article  CAS  PubMed  Google Scholar 

  21. Corlu A, Loyer P (2012) Regulation of the G1/S transition in hepatocytes: involvement of the cyclin-dependent kinase Cdk1 in the DNA replication. Int J Hepatol 2012:689324

    Article  PubMed Central  PubMed  Google Scholar 

  22. McGowan JA (1986) Hepatocyte proliferation in culture. In: Guillouzo A, Guillouzo C (eds) Isolated and cultured hepatocytes. INSERM, Paris, pp 13–38

    Google Scholar 

  23. Albrecht JH, Hu MY, Cerra FB (1995) Distinct patterns of cyclin D1 regulation in models of liver regeneration and human liver. Biochem Biophys Res Commun 209:648–655

    Article  CAS  PubMed  Google Scholar 

  24. Isom HC, Secott T, Georgoff I et al (1985) Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci U S A 82:3252–3256

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Guguen-Guillouzo C, Clement B, Baffet G et al (1983) Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp Cell Res 143:47–54

    Article  CAS  PubMed  Google Scholar 

  26. Waxman DJ, Morrissey JJ, Naik S et al (1990) Phenobarbital induction of cytochromes P450: high-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response. Biochem J 271:113–119

    CAS  PubMed Central  PubMed  Google Scholar 

  27. Bissell DM, Arenson DM, Maher JJ et al (1987) Support of cultured hepatocytes by a laminin-rich gel: evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest 79:801–812

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Kojima T, Mitaka T, Paul DL et al (1995) Reappearance and long-term maintenance of connexin32 in proliferated adult rat hepatocytes: use of serum-free L-15 medium supplemented with EGF and DMSO. J Cell Sci 108:1347–1357

    CAS  PubMed  Google Scholar 

  29. Corlu A, Ilyin G, Cariou S et al (1997) The coculture: a system for studying the regulation of liver differentiation/proliferation activity and its control. Cell Biol Toxicol 13:235–242

    Article  CAS  PubMed  Google Scholar 

  30. Hansen LK, Albrecht JH (1999) Regulation of the hepatocyte cell cycle by type I collagen matrix: role of cyclin D1. J Cell Sci 112:2971–2981

    CAS  PubMed  Google Scholar 

  31. De Smet K, Loyer P, Gilot D et al (2001) Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes. Biochem Pharmacol 61:1293–1303

    Article  PubMed  Google Scholar 

  32. Corlu A, Kneip B, Lhadi C et al (1991) A plasma membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes. J Cell Biol 115:505–515

    Article  CAS  PubMed  Google Scholar 

  33. Serandour AL, Loyer P, Garnier D et al (2005) TNFalpha-mediated extracellular matrix remodeling is required for multiple division cycles in rat hepatocytes. Hepatology 41:478–486

    Article  CAS  PubMed  Google Scholar 

  34. Gripon P, Rumin S, Urban S et al (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99:15655–15660

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Cerec V, Glaise D, Garnier D et al (2007) Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 45:957–967

    Article  CAS  PubMed  Google Scholar 

  36. Aninat C, Piton A, Glaise D et al (2006) Expression of cytochrome P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos 34:75–83

    Article  CAS  PubMed  Google Scholar 

  37. Kang LI, Mars WM, Michalopoulos GK (2012) Signals and cells involved in regulating liver regeneration. Cells 1:1261–1292

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Giandomenico AR, Cerniglia GE, Biaglow JE et al (1997) The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide. Free Radic Biol Med 23:426–434

    Article  CAS  PubMed  Google Scholar 

  39. Williams GM, Gunn JM (1974) Long-term cell culture of rat liver epithelial cells. Exp Cell Res 89:139–142

    Article  CAS  PubMed  Google Scholar 

  40. Williams GM, Weisburger EK, Weisburger JH (1971) Isolation and long-term cell culture of epithelial-like cells from rat liver. Exp Cell Res 69:106–112

    Article  CAS  PubMed  Google Scholar 

  41. Deschatrette J, Weiss MC (1974) Characteri-zation of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie 56:1603–1611

    Article  CAS  PubMed  Google Scholar 

  42. Bailly-Maitre B, de Sousa G, Zucchini N et al (2002) Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone. Cell Death Differ 9:945–955

    Article  CAS  PubMed  Google Scholar 

  43. Gilot D, Loyer P, Corlu A et al (2002) Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione S-transferase regulation. J Biol Chem 77:49220–49229

    Article  Google Scholar 

  44. Gerbal-Chaloin S, Duret C, Raulet E et al (2010) Isolation and culture of adult human liver progenitor cells: in vitro differentiation to hepatocyte-like cells. Methods Mol Biol 640:247–260

    Article  CAS  PubMed  Google Scholar 

  45. Blanc P, Etienne H, Daujat M et al (1992) Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor alpha, and human serum. Gastroenterology 102:1340–1350

    CAS  PubMed  Google Scholar 

  46. Reddy CC, Wells A, Lauffenburger DA (1996) Receptor-mediated effects on ligand availability influence relative mitogenic potencies of epidermal growth factor and transforming growth factor alpha. J Cell Physiol 166:512–522

    Article  CAS  PubMed  Google Scholar 

  47. Takatsuki K, Fujiwara K, Hayashi S et al (1990) Acceleration of DNA synthesis in post-hepatectomised regenerating liver of normal rat by insulin and glucagon. Life Sci 29:2609–2615

    Article  Google Scholar 

  48. McMahon JB, Richards WL, del Campo AA et al (1986) Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture. Cancer Res 46:4665–4671

    CAS  PubMed  Google Scholar 

  49. Berasain C, Garcia-Trevijano ER, Castillo J et al (2005) Amphiregulin: an early trigger of liver regeneration in mice. Gastroenterology 128:424–432

    Article  CAS  PubMed  Google Scholar 

  50. Ito N, Kawata S, Tamura S et al (1994) Heparin-binding EGF-like growth factor is a potent mitogen for rat hepatocytes. Biochem Biophys Res Commun 198:25–31

    Article  CAS  PubMed  Google Scholar 

  51. Cruise JL, Knechtle SJ, Bollinger RR et al (1987) Alpha 1-adrenergic effects and liver regeneration. Hepatology 7:1189–1194

    Article  CAS  PubMed  Google Scholar 

  52. Lesurtel M, Graf R, Aleil B et al (2006) Platelet-derived serotonin mediates liver regeneration. Science 312:104–107

    Article  CAS  PubMed  Google Scholar 

  53. Sharon Goh YP, Henderson NC, Heredia JE et al (2013) Eosinophils secrete IL-4 to facilitate liver regeneration. Proc Natl Acad Sci U S A 110:9914–9919

    Article  Google Scholar 

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Authors and Affiliations

  1. Inserm, UMR 991, Liver, Metabolisms and Cancer, Hôpital Pontchaillou, University of Rennes 1, Rennes Cedex, 35033, France

    Anne Corlu & Pascal Loyer

Authors
  1. Anne Corlu
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  2. Pascal Loyer
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Correspondence to Pascal Loyer .

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Editors and Affiliations

  1. Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium

    Mathieu Vinken

  2. Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium

    Vera Rogiers

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Corlu, A., Loyer, P. (2015). Culture Conditions Promoting Hepatocyte Proliferation and Cell Cycle Synchronization. In: Vinken, M., Rogiers, V. (eds) Protocols in In Vitro Hepatocyte Research. Methods in Molecular Biology, vol 1250. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2074-7_3

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  • DOI: https://doi.org/10.1007/978-1-4939-2074-7_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2073-0

  • Online ISBN: 978-1-4939-2074-7

  • eBook Packages: Springer Protocols

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