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Hepatocyte Differentiation

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Hepatocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 640))

Abstract

Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen “sandwich” system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

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References

  1. Perkins, E.J., Bao, W., Guan, X., Ang, C.Y., Wolfinger, R.D., Chu, T.M., Meyer, S.A., and Inouye, L.S. (2006) Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine. BMC Bioinform. 7 Suppl 4, S22.

    Google Scholar 

  2. Berry, M.N. and Friend, D.S. (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43, 506–520.

    PubMed  CAS  Google Scholar 

  3. Seglen, P.O. (1976) Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83.

    PubMed  CAS  Google Scholar 

  4. Guguen-Guillouzo, C., Campion, J.P., Brissot, P., Glaise, D., Launois, B., Bourel, M., and Guillouzo, A. (1982) High yield preparation of isolated human adult hepatocytes by enzymatic perfusion of the liver. Cell Biol. Int. Rep. 6, 625–628.

    PubMed  CAS  Google Scholar 

  5. Reese, J.A. and Byard, J.L. (1981) Isolation and culture of adult hepatocytes from liver biopsies. In Vitro 17, 935–940.

    PubMed  CAS  Google Scholar 

  6. Strom, S.C., Jirtle, R.L., Jones, R.S., Novicki, D.L., Rosenberg, M.R., Novotny, A., Irons, G., McLain, J.R., and Michalopoulos, G. (1982) Isolation, culture, and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68, 771–778.

    PubMed  CAS  Google Scholar 

  7. Clayton, D.F., and Darnell, J.E., Jr. (1983) Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol. Cell Biol. 3, 1552–1561.

    PubMed  CAS  Google Scholar 

  8. Guzelian, P.S., Bissell, D.M., and Meyer, U.A. (1977) Drug metabolism in adult rat hepatocytes in primary monolayer culture. Gastroenterology 72, 1232–1239.

    PubMed  CAS  Google Scholar 

  9. Maslansky, C.J. and Williams, G.M. (1982) Primary cultures and the levels of cytochrome P450 in hepatocytes from mouse, rat, hamster, and rabbit liver. In Vitro 18, 683–693.

    PubMed  CAS  Google Scholar 

  10. Loyer, P., Cariou, S., Glaise, D., Bilodeau, M., Baffet, G., and Guguen-Guillouzo, C. (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.

    PubMed  CAS  Google Scholar 

  11. Rana, B., Mischoulon, D., Xie, Y., Bucher, N.L., and Farmer, S.R. (1994) Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. Mol. Cell Biol. 14, 5858–5869.

    PubMed  CAS  Google Scholar 

  12. Block, G.D., Locker, J., Bowen, W.C., Petersen, B.E., Katyal, S., Strom, S.C., Riley, T., Howard, T.A., and Michalopoulos, G.K. (1996) Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J. Cell Biol. 132, 1133–1149.

    PubMed  CAS  Google Scholar 

  13. Sidhu, J.S., Liu, F., and Omiecinski, C.J. (2004) Phenobarbital responsiveness as a uniquely sensitive indicator of hepatocyte differentiation status: requirement of dexamethasone and extracellular matrix in establishing the functional integrity of cultured primary rat hepatocytes. Exp. Cell Res. 292, 252–264.

    PubMed  CAS  Google Scholar 

  14. Paine, A.J. and Andreakos, E. (2004) Activation of signalling pathways during hepatocyte isolation: relevance to toxicology in vitro. Toxicol. In Vitro 18, 187–193.

    PubMed  CAS  Google Scholar 

  15. Suzuki, A., Iwama, A., Miyashita, H., Nakauchi, H., and Taniguchi, H. (2003) Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells. Development 130, 2513–2524.

    PubMed  CAS  Google Scholar 

  16. Runge, D., Runge, D.M., Bowen, W.C., Locker, J., and Michalopoulos, G.K. (1997) Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins. Biol. Chem. 378, 873–881.

    PubMed  CAS  Google Scholar 

  17. Thomas, R.J., Bhandari, R., Barrett, D.A., Bennett, A.J., Fry, J.R., Powe, D., Thomson, B.J., and Shakesheff, K.M. (2005) The effect of three-dimensional co-culture of hepatocytes and hepatic stellate cells on key hepatocyte functions in vitro. Cells Tissues Organs 181, 67–79.

    PubMed  Google Scholar 

  18. Tzanakakis, E.S., Waxman, D.J., Hansen, L.K., Remmel, R.P., and Hu, W.S. (2002) Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer. Cell Biol. Toxicol. 18, 13–27.

    PubMed  CAS  Google Scholar 

  19. Nahmias, Y., Casali, M., Barbe, L., Berthiaume, F., and Yarmush, M.L. (2006) Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes. Hepatology 43, 257–265.

    PubMed  CAS  Google Scholar 

  20. Williams, S.N., Callies, R.M., and Brindle, K.M. (1997) Mapping of oxygen tension and cell distribution in a hollow-fiber bioreactor using magnetic resonance imaging. Biotechnol. Bioeng. 56, 56–61.

    PubMed  CAS  Google Scholar 

  21. Nyberg, S.L., Shatford, R.A., Peshwa, M.V., White, J.G., Cerra, F.B., and Hu, W.S. (1993) Evaluation of a hepatocyte-entrapment hollow fiber bioreactor: a potential bioartificial liver. Biotechnol. Bioeng. 41, 194–203.

    PubMed  CAS  Google Scholar 

  22. Li, A.P., Barker, G., Beck, D., Colburn, S., Monsell, R., and Pellegrin, C. (1993) Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver. In Vitro Cell Dev. Biol. 29A, 249–254.

    PubMed  CAS  Google Scholar 

  23. De Bartolo, L., Morelli, S., Rende, M., Campana, C., Salerno, S., Quintiero, N., and Drioli, E. (2007) Human hepatocyte morphology and functions in a multibore fiber bioreactor. Macromol. Biosci. 7, 671–680.

    PubMed  Google Scholar 

  24. Powers, M.J., Domansky, K., Kaazempur-Mofrad, M.R., Kalezi, A., Capitano, A., Upadhyaya, A., Kurzawski, P., Wack, K.E., Stolz, D.B., Kamm, R., and Griffith, L.G. (2002) A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 78, 257–269.

    PubMed  CAS  Google Scholar 

  25. De Bartolo, L., Salerno, S., Morelli, S., Giorno, L., Rende, M., Memoli, B., Procino, A., Andreucci, V.E., Bader, A., and Drioli, E. (2006) Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor. Biomaterials 27, 4794–4803.

    PubMed  Google Scholar 

  26. Flendrig, L.M., la Soe, J.W., Jorning, G.G., Steenbeek, A., Karlsen, O.T., Bovee, W.M., Ladiges, N.C., te Velde, A.A., and Chamuleau, R.A. (1997) In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates. J. Hepatol. 26, 1379–1392.

    PubMed  CAS  Google Scholar 

  27. Lu, H.F., Lim, W.S., Zhang, P.C., Chia, S.M., Yu, H., Mao, H.Q., and Leong, K.W. (2005) Galactosylated poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture. Tissue Eng. 11, 1667–1677.

    PubMed  CAS  Google Scholar 

  28. Pless, G., Steffen, I., Zeilinger, K., Sauer, I.M., Katenz, E., Kehr, D.C., Roth, S., Mieder, T., Schwartlander, R., Muller, C., Wegner, B., Hout, M.S., and Gerlach, J.C. (2006) Evaluation of primary human liver cells in bioreactor cultures for extracorporeal liver support on the basis of urea production. Artif. Organs 30, 686–694.

    PubMed  CAS  Google Scholar 

  29. MacDonald, J.M., Wolfe, S.P., Roy-Chowdhury, I., Kubota, H., and Reid, L.M. (2001) Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver. Ann. NY Acad. Sci. 944, 334–343.

    PubMed  CAS  Google Scholar 

  30. Kan, P., Miyoshi, H., Yanagi, K., and Ohshima, N. (1998) Effects of shear stress on metabolic function of the co-culture system of hepatocyte/nonparenchymal cells for a bioartificial liver. ASAIO J. 44, M441–M444.

    PubMed  CAS  Google Scholar 

  31. Mufti, N.A., Bleckwenn, N.A., Babish, J.G., and Shuler, M.L. (1995) Possible involvement of the Ah receptor in the induction of cytochrome P-450IA1 under conditions of hydrodynamic shear in microcarrier-attached hepatoma cell lines. Biochem. Biophys. Res. Commun. 208, 144–152.

    PubMed  CAS  Google Scholar 

  32. Miyazawa, M., Torii, T., Toshimitsu, Y., Okada, K., and Koyama, I. (2007) Hepatocyte dynamics in a three-dimensional rotating bioreactor. J. Gastroenterol. Hepatol. 22, 1959–1964.

    PubMed  CAS  Google Scholar 

  33. Kono, Y., Yang, S., and Roberts, E.A. (1997) Extended primary culture of human hepatocytes in a collagen gel sandwich system. In Vitro Cell Dev. Biol. Anim. 33, 467–472.

    PubMed  CAS  Google Scholar 

  34. Nussler, A.K., Wang, A., Neuhaus, P., Fischer, J., Yuan, J., Liu, L., Zeilinger, K., Gerlach, J., Arnold, P.J., and Albrecht, W. (2001) The suitability of hepatocyte culture models to study various aspects of drug metabolism. ALTEX 18, 91–101.

    PubMed  CAS  Google Scholar 

  35. Ezzell, R.M., Toner, M., Hendricks, K., Dunn, J.C., Tompkins, R.G., and Yarmush, M.L. (1993) Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes. Exp. Cell Res. 208, 442–452.

    PubMed  CAS  Google Scholar 

  36. Sidhu, J.S., Farin, F.M., and Omiecinski, C.J. (1993) Influence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1, 2B2, and 3A1 genes in primary adult rat hepatocyte culture. Arch. Biochem. Biophys. 301, 103–113.

    PubMed  CAS  Google Scholar 

  37. Tuschl, G. and Mueller, S.O. (2006) Effects of cell culture conditions on primary rat hepatocytes-cell morphology and differential gene expression. Toxicology 218, 205–215.

    PubMed  CAS  Google Scholar 

  38. Annaert, P.P., Brouwer, K.L. (2005) Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 33, 388–394.

    PubMed  CAS  Google Scholar 

  39. Liu, X., LeCluyse, E.L., Brouwer, K.R., Gan, L.S., Lemasters, J.J., Stieger, B., Meier, P.J., and Brouwer, K.L. (1999) Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am. J. Physiol. 277, G12–G21.

    PubMed  CAS  Google Scholar 

  40. Moghe, P.V., Berthiaume, F., Ezzell, R.M., Toner, M., Tompkins, R.G., and Yarmush, M.L. (1996) Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. Biomaterials 17, 373–385.

    PubMed  CAS  Google Scholar 

  41. Kern, A., Bader, A., Pichlmayr, R., and Sewing, K.F. (1997) Drug metabolism in hepatocyte sandwich cultures of rats and humans. Biochem. Pharmacol. 54, 761–772.

    PubMed  CAS  Google Scholar 

  42. Madan, A., Graham, R.A., Carroll, K.M., Mudra, D.R., Burton, L.A., Krueger, L.A., Downey, A.D., Czerwinski, M., Forster, J., Ribadeneira, M.D., Gan, L.S., LeCluyse, E.L., Zech, K., Robertson, P., Jr., Koch, P., Antonian, L., Wagner, G., Yu, L., and Parkinson, A. (2003) Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab. Dispos. 31, 421–431.

    PubMed  CAS  Google Scholar 

  43. Mingoia, R.T., Nabb, D.L., Yang, C.H., and Han, X. (2007) Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening. Toxicol. In Vitro 21, 165–173.

    PubMed  CAS  Google Scholar 

  44. Ben Ze’ev, A., Robinson, G.S., Bucher, N.L., and Farmer, S.R. (1988) Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc. Natl. Acad. Sci. USA 85, 2161–2165.

    PubMed  Google Scholar 

  45. Nagaki, M., Shidoji, Y., Yamada, Y., Sugiyama, A., Tanaka, M., Akaike, T., Ohnishi, H., Moriwaki, H., and Muto, Y. (1995) Regulation of hepatic genes and liver transcription factors in rat hepatocytes by extracellular matrix. Biochem. Biophys. Res. Commun. 210, 38–43.

    PubMed  CAS  Google Scholar 

  46. Kimata, T., Nagaki, M., Tsukada, Y., Ogiso, T., and Moriwaki, H. (2006) Hepatocyte nuclear factor-4alpha and -1 small interfering RNA inhibits hepatocyte differentiation induced by extracellular matrix. Hepatol. Res. 35, 3–9.

    PubMed  CAS  Google Scholar 

  47. DiPersio, C.M., Jackson, D.A., and Zaret, K.S. (1991) The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology. Mol. Cell Biol. 11, 4405–4414.

    PubMed  CAS  Google Scholar 

  48. Caron, J.M. (1990) Induction of albumin gene transcription in hepatocytes by extracellular matrix proteins. Mol. Cell Biol. 10, 1239–1243.

    PubMed  CAS  Google Scholar 

  49. DiPersio, C.M., Jackson, D.A., and Zaret, K.S. (1991) The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology. Mol. Cell Biol. 11, 4405–4414.

    PubMed  CAS  Google Scholar 

  50. Juliano, R.L. and Haskill, S. (1993) Signal transduction from the extracellular matrix. J. Cell Biol. 120, 577–585.

    PubMed  CAS  Google Scholar 

  51. Gullberg, D., Turner, D.C., Borg, T.K., Terracio, L., and Rubin, K. (1990) Different beta 1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts. Exp. Cell Res. 190, 254–264.

    PubMed  CAS  Google Scholar 

  52. Gullberg, D., Gehlsen, K.R., Turner, D.C., Ahlen, K., Zijenah, L.S., Barnes, M.J., and Rubin, K. (1992) Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell – collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I. EMBO J. 11, 3865–3873.

    PubMed  CAS  Google Scholar 

  53. Johansson, S., Forsberg, E., and Lundgren, B. (1987) Comparison of fibronectin receptors from rat hepatocytes and fibroblasts. J. Biol. Chem. 262, 7819–7824.

    PubMed  CAS  Google Scholar 

  54. Lora, J.M., Rowader, K.E., Soares, L., Giancotti, F., and Zaret, K.S. (1998) Alpha3beta1-integrin as a critical mediator of the hepatic differentiation response to the extracellular matrix. Hepatology 28, 1095–1104.

    PubMed  CAS  Google Scholar 

  55. Gkretsi, V., Apte, U., Mars, W.M., Bowen, W.C., Luo, J.H., Yang, Y., Yu, Y.P., Orr, A., St Arnaud, R., Dedhar, S., Kaestner, K.H., Wu, C., and Michalopoulos, G.K. (2008) Liver-specific ablation of integrin-linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly. Hepatology 48, 1932–1941.

    PubMed  Google Scholar 

  56. Bresnick, E.H., Dalman, F.C., Sanchez, E.R., and Pratt, W.B. (1989) Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J. Biol. Chem. 264, 4992–4997.

    PubMed  CAS  Google Scholar 

  57. Johnson, J.L. and Toft, D.O. (1995) Binding of p23 and hsp90 during assembly with the progesterone receptor. Mol. Endocrinol. 9, 670–678.

    PubMed  CAS  Google Scholar 

  58. Davies, T.H., Ning, Y.M., and Sanchez, E.R. (2002) A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins. J. Biol. Chem. 277, 4597–4600.

    PubMed  CAS  Google Scholar 

  59. Smith, D.F. (2004) Tetratricopeptide repeat cochaperones in steroid receptor complexes. Cell Stress Chaperones 9, 109–121.

    PubMed  CAS  Google Scholar 

  60. Glass, C.K. and Rosenfeld, M.G. (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121–141.

    PubMed  CAS  Google Scholar 

  61. Freedman, N.D. and Yamamoto, K.R. (2004) Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor. Mol. Biol. Cell 15, 2276–2286.

    PubMed  CAS  Google Scholar 

  62. Garside, H., Stevens, A., Farrow, S., Normand, C., Houle, B., Berry, A., Maschera, B., and Ray, D. (2004) Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain. J. Biol. Chem. 279, 50050–50059.

    PubMed  CAS  Google Scholar 

  63. Widen, C., Gustafsson, J.A., and Wikstrom, A.C. (2003) Cytosolic glucocorticoid receptor interaction with nuclear factor-kappa B proteins in rat liver cells. Biochem. J. 373, 211–220.

    PubMed  CAS  Google Scholar 

  64. Nelson, G., Wilde, G.J., Spiller, D.G., Kennedy, S.M., Ray, D.W., Sullivan, E., Unitt, J.F., and White, M.R. (2003) NF-kappaB signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms. J. Cell Sci. 116, 2495–2503.

    PubMed  CAS  Google Scholar 

  65. Li, W.C., Ralphs, K.L., Slack, J.M., and Tosh, D. (2007) Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors. Int. J. Biochem. Cell Biol. 39, 541–554.

    PubMed  CAS  Google Scholar 

  66. Dasgupta, A., Hughey, R., Lancin, P., Larue, L., and Moghe, P.V. (2005) E-cadherin synergistically induces hepatospecific phenotype and maturation of embryonic stem cells in conjunction with hepatotrophic factors. Biotechnol. Bioeng. 92, 257–266.

    PubMed  CAS  Google Scholar 

  67. Scheving, L.A., Stevenson, M.C., Zhang, X., and Russell, W.E. (2008) Cultured rat hepatocytes upregulate Akt and ERK in an ErbB-2-dependent manner. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G322–G331.

    PubMed  CAS  Google Scholar 

  68. Scheving, L.A., Buchanan, R., Krause, M.A., Zhang, X., Stevenson, M.C., and Russell, W.E. (2007) Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 293, G552–G559.

    PubMed  CAS  Google Scholar 

  69. Baharvand, H., Hashemi, S.M., and Shahsavani, M. (2008) Differentiation of human embryonic stem cells into functional hepatocyte-like cells in a serum-free adherent culture condition. Differentiation 76, 465–477.

    PubMed  CAS  Google Scholar 

  70. Miki, R., Tatsumi, N., Matsumoto, K., and Yokouchi, Y. (2008) New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts. Am. J. Physiol. Gastrointest. Liver Physiol. 294, G529–G539.

    PubMed  CAS  Google Scholar 

  71. Saito, K., Yoshikawa, M., Ouji, Y., Moriya, K., Nishiofuku, M., Ueda, S., Hayashi, N., Ishizaka, S., and Fukui, H. (2006) Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells. World J. Gastroenterol. 12, 6818–6827.

    PubMed  CAS  Google Scholar 

  72. Shirahashi, H., Wu, J., Yamamoto, N., Catana, A., Wege, H., Wager, B., Okita, K., and Zern, M.A. (2004) Differentiation of human and mouse embryonic stem cells along a hepatocyte lineage. Cell Transplant. 13, 197–211.

    PubMed  Google Scholar 

  73. Page, J.L., Johnson, M.C., Olsavsky, K.M., Strom, S.C., Zarbl, H., and Omiecinski, C.J. (2007) Gene expression profiling of extracellular matrix as an effector of human hepatocyte phenotype in primary cell culture. Toxicol. Sci. 97, 384–397.

    PubMed  CAS  Google Scholar 

  74. Strom, S.C., Pisarov, L.A., Dorko, K., Thompson, M.T., Schuetz, J.D., and Schuetz, E.G. (1996) Use of human hepatocytes to study P450 gene induction. Methods Enzymol. 272, 388–401.

    PubMed  CAS  Google Scholar 

  75. Zegers, M.M. and Hoekstra, D. (1998) Mechanisms and functional features of polarized membrane traffic in epithelial and hepatic cells. Biochem. J. 336 (Pt 2), 257–269.

    PubMed  CAS  Google Scholar 

  76. Hoffmaster, K.A., Turncliff, R.Z., LeCluyse, E.L., Kim, R.B., Meier, P.J., and Brouwer, K.L. (2004) P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm. Res. 21, 1294–1302.

    PubMed  CAS  Google Scholar 

  77. 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.

    PubMed  CAS  Google Scholar 

  78. Kojima, T., Mitaka, T., Paul, D.L., Mori, M., and Mochizuki, Y. (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 (Pt 4), 1347–1357.

    PubMed  CAS  Google Scholar 

  79. Stoehr, S.A. and Isom, H.C. (2003) Gap junction-mediated intercellular communication in a long-term primary mouse hepatocyte culture system. Hepatology 38, 1125–1135.

    PubMed  Google Scholar 

  80. Agarwal, S., Holton, K.L., and Lanza, R. (2008) Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells 26, 1117–1127.

    PubMed  CAS  Google Scholar 

  81. Liu, X., Brouwer, K.L., Gan, L.S., Brouwer, K.R., Stieger, B., Meier, P.J., Audus, K.L., and LeCluyse, E.L. (1998) Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration. Pharm. Res. 15, 1533–1539.

    PubMed  CAS  Google Scholar 

  82. Chandra, P., LeCluyse, E.L., and Brouwer, K.L. (2001) Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes. In Vitro Cell Dev. Biol. Anim. 37, 380–385.

    PubMed  CAS  Google Scholar 

  83. LeCluyse, E., Bullock, P., Madan, A., Carroll, K., and Parkinson, A. (1999) Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes. Drug Metab. Dispos. 27, 909–915.

    PubMed  CAS  Google Scholar 

  84. Runge, D., Runge, D.M., Jager, D., Lubecki, K.A., Beer, S.D., Karathanasis, S., Kietzmann, T., Strom, S.C., Jungermann, K., Fleig, W.E., and Michalopoulos, G.K. (2000) Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem. Biophys. Res. Commun. 269, 46–53.

    PubMed  CAS  Google Scholar 

  85. Rambhatla, L., Chiu, C.P., Kundu, P., Peng, Y., and Carpenter, M.K. (2003) Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplant. 12, 1–11.

    PubMed  Google Scholar 

  86. Yamamoto, Y., Teratani, T., Yamamoto, H., Quinn, G., Murata, S., Ikeda, R., Kinoshita, K., Matsubara, K., Kato, T., and Ochiya, T. (2005) Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells. Hepatology 42, 558–567.

    PubMed  CAS  Google Scholar 

  87. Hay, D.C., Zhao, D., Fletcher, J., Hewitt, Z.A., McLean, D., Urruticoechea-Uriguen, A., Black, J.R., Elcombe, C., Ross, J.A., Wolf, R., and Cui, W. (2008) Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells 26, 894–902.

    PubMed  CAS  Google Scholar 

  88. Olsavsky, K.M., Page, J.L., Johnson, M.C., Zarbl, H., Strom, S.C., and Omiecinski, C.J. (2007) Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues. Toxicol. Appl. Pharmacol. 222, 42–56.

    PubMed  CAS  Google Scholar 

  89. Farkas, D. and Tannenbaum, S.R. (2005) Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes. Toxicol. Sci. 85, 927–934.

    PubMed  CAS  Google Scholar 

  90. Hino, H., Tateno, C., Sato, H., Yamasaki, C., Katayama, S., Kohashi, T., Aratani, A., Asahara, T., Dohi, K., and Yoshizato, K. (1999) A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes. Biochem. Biophys. Res. Commun. 256, 184–191.

    PubMed  CAS  Google Scholar 

  91. Ferrini, J.B., Pichard, L., Domergue, J., and Maurel, P. (1997) Long-term primary cultures of adult human hepatocytes. Chem. Biol. Interact. 107, 31–45.

    PubMed  CAS  Google Scholar 

  92. Miller, L.L., Bly, C.G., Watson, M.L., and Bale, W.F. (1951) The dominant role of the liver in plasma protein synthesis; a direct study of the isolated perfused rat liver with the aid of lysine-epsilon-C14. J. Exp. Med. 94, 431–453.

    PubMed  CAS  Google Scholar 

  93. Feldmann, G., Scoazec, J.Y., Racine, L., and Bernuau, D. (1992) Functional hepatocellular heterogeneity for the production of plasma proteins. Enzyme 46, 139–154.

    PubMed  CAS  Google Scholar 

  94. Ali-Osman, F., Brunner, J.M., Kutluk, T.M., and Hess, K. (1997) Prognostic significance of glutathione S-transferase pi expression and subcellular localization in human gliomas. Clin. Cancer Res. 3, 2253–2261.

    PubMed  CAS  Google Scholar 

  95. Lavon, N. and Benvenisty, N. (2005) Study of hepatocyte differentiation using embryonic stem cells. J. Cell Biochem. 96, 1193–1202.

    PubMed  CAS  Google Scholar 

  96. Kim, H.M., Han, S.B., Hyun, B.H., Ahn, C.J., Cha, Y.N., Jeong, K.S., and Oh, G.T. (1995) Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions. J. Toxicol. Sci. 20, 565–578.

    PubMed  CAS  Google Scholar 

  97. Takagi, M., Kojima, N., and Yoshida, T. (2000) Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7. Cytotechnology 32, 9–15.

    PubMed  CAS  Google Scholar 

  98. Haussinger, D. and Schliess, F. (2007) Glutamine metabolism and signaling in the liver. Front. Biosci. 12, 371–391.

    PubMed  Google Scholar 

  99. Handschin, C. and Meyer, U.A. (2003) Induction of drug metabolism: the role of nuclear receptors. Pharmacol. Rev. 55, 649–673.

    PubMed  CAS  Google Scholar 

  100. LeCluyse, E.L. (2001) Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur. J. Pharm. Sci. 13, 343–368.

    PubMed  CAS  Google Scholar 

  101. Gerbal-Chaloin, S., Daujat, M., Pascussi, J.M., Pichard-Garcia, L., Vilarem, M.J., and Maurel, P. (2002) Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor. J. Biol. Chem. 277, 209–217.

    PubMed  CAS  Google Scholar 

  102. Pascussi, J.M., Gerbal-Chaloin, S., Fabre, J.M., Maurel, P., and Vilarem, M.J. (2000) Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. Mol. Pharmacol. 58, 1441–1450.

    PubMed  CAS  Google Scholar 

  103. Whysner, J., Ross, P.M., and Williams, G.M. (1996) Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion. Pharmacol. Ther. 71, 153–191.

    PubMed  CAS  Google Scholar 

  104. Sanders, S. and Thorgeirsson, S.S. (1999) Phenobarbital promotes liver growth in c-myc/TGF-alpha transgenic mice by inducing hypertrophy and inhibiting apoptosis. Carcinogenesis 20, 41–49.

    PubMed  CAS  Google Scholar 

  105. Calvisi, D.F., Ladu, S., Factor, V.M., and Thorgeirsson, S.S. (2004) Activation of beta-catenin provides proliferative and invasive advantages in c-myc/TGF-alpha hepatocarcinogenesis promoted by phenobarbital. Carcinogenesis 25, 901–908.

    PubMed  CAS  Google Scholar 

  106. Mansbach, J.M., Mills, J.J., Boyer, I.J., De Souza, A.T., Hankins, G.R., and Jirtle, R.L. (1996) Phenobarbital selectively promotes initiated cells with reduced TGF beta receptor levels. Carcinogenesis 17, 171–174.

    PubMed  CAS  Google Scholar 

  107. Gonzales, A.J., Christensen, J.G., Preston, R.J., Goldsworthy, T.L., Tlsty, T.D., and Fox, T.R. (1998) Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital. Carcinogenesis 19, 1173–1183.

    PubMed  CAS  Google Scholar 

  108. Phillips, J.M., Yamamoto, Y., Negishi, M., Maronpot, R.R., and Goodman, J.I. (2007) Orphan nuclear receptor constitutive active/androstane receptor-mediated alterations in DNA methylation during phenobarbital promotion of liver tumorigenesis. Toxicol. Sci. 96, 72–82.

    PubMed  CAS  Google Scholar 

  109. Kakizaki, S., Yamamoto, Y., Ueda, A., Moore, R., Sueyoshi, T., and Negishi, M. (2003) Phenobarbital induction of drug/steroid-metabolizing enzymes and nuclear receptor CAR. Biochim. Biophys. Acta. 1619, 239–242.

    PubMed  CAS  Google Scholar 

  110. Kodama, S. and Negishi, M. (2006) Phenobarbital confers its diverse effects by activating the orphan nuclear receptor car. Drug Metab. Rev. 38, 75–87.

    PubMed  CAS  Google Scholar 

  111. Qatanani, M. and Moore, D.D. (2005) CAR, the continuously advancing receptor, in drug metabolism and disease. Curr. Drug Metab. 6, 329–339.

    PubMed  CAS  Google Scholar 

  112. Yamamoto, Y., Moore, R., Goldsworthy, T.L., Negishi, M., and Maronpot, R.R. (2004) The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice. Cancer Res. 64, 7197–7200.

    PubMed  CAS  Google Scholar 

  113. Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K., and Negishi, M. (1999) Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol. Cell. Biol. 19, 6318–6322.

    PubMed  CAS  Google Scholar 

  114. Kobayashi, K., Sueyoshi, T., Inoue, K., Moore, R., and Negishi, M. (2003) Cytoplasmic accumulation of the nuclear receptor CAR by a tetratricopeptide repeat protein in HepG2 cells. Mol. Pharmacol. 64, 1069–1075.

    PubMed  CAS  Google Scholar 

  115. Yoshinari, K., Kobayashi, K., Moore, R., Kawamoto, T., and Negishi, M. (2003) Identification of the nuclear receptor CAR: HSP90 complex in mouse liver and recruitment of protein phosphatase 2A in response to phenobarbital. FEBS Lett. 548, 17–20.

    PubMed  CAS  Google Scholar 

  116. Zelko, I., Sueyoshi, T., Kawamoto, T., Moore, R., and Negishi, M. (2001) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver. Mol. Cell Biol. 21, 2838–2846.

    PubMed  CAS  Google Scholar 

  117. Frank, C., Gonzalez, M.M., Oinonen, C., Dunlop, T.W., and Carlberg, C. (2003) Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor. J. Biol. Chem. 278, 43299–43310.

    PubMed  CAS  Google Scholar 

  118. Muangmoonchai, R., Smirlis, D., Wong, S.C., Edwards, M., Phillips, I.R., and Shephard, E.A. (2001) Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) is mediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires steroid co-activator 1 (SRC-1) and the transcription factor Sp1. Biochem. J. 355, 71–78.

    PubMed  CAS  Google Scholar 

  119. Min, G., Kemper, J.K., and Kemper, B. (2002) Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo. J. Biol. Chem. 277, 26356–26363.

    PubMed  CAS  Google Scholar 

  120. Shiraki, T., Sakai, N., Kanaya, E., and Jingami, H. (2003) Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state. J. Biol. Chem. 278, 11344–11350.

    PubMed  CAS  Google Scholar 

  121. Honkakoski, P. and Negishi, M. (1997) Characterization of a phenobarbital-responsive enhancer module in mouse P450 Cyp2b10 gene. J. Biol. Chem. 272, 14943–14949.

    PubMed  CAS  Google Scholar 

  122. Ramsden, R., Beck, N.B., Sommer, K.M., and Omiecinski, C.J. (1999) Phenobarbital responsiveness conferred by the 5’-flanking region of the rat CYP2B2 gene in transgenic mice. Gene 228, 169–179.

    PubMed  CAS  Google Scholar 

  123. LeCluyse, E., Madan, A., Hamilton, G., Carroll, K., DeHaan, R., and Parkinson, A. (2000) Expression and regulation of cytochrome P450 enzymes in primary cultures of human hepatocytes. J. Biochem. Mol. Toxicol. 14, 177–188.

    PubMed  CAS  Google Scholar 

  124. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Center for Molecular Toxicology & Carcinogenesis and Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA, USA

    Katy M. Olsavsky Goyak, Elizabeth M. Laurenzana & Curtis J. Omiecinski

  2. ExxonMobil Biomedical Sciences, Inc., Annandale, NJ, USA

    Katy M. Olsavsky Goyak

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  1. Katy M. Olsavsky Goyak
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  2. Elizabeth M. Laurenzana
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  3. Curtis J. Omiecinski
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Correspondence to Katy M. Olsavsky Goyak .

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  1. INSERM U632, Labo. Physiopathologie Hépatique, Université Montpellier I, rte. Mende 1919, Montpellier, 34293, France

    Patrick Maurel

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Olsavsky Goyak, K.M., Laurenzana, E.M., Omiecinski, C.J. (2010). Hepatocyte Differentiation. In: Maurel, P. (eds) Hepatocytes. Methods in Molecular Biology, vol 640. Humana Press. https://doi.org/10.1007/978-1-60761-688-7_6

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  • DOI: https://doi.org/10.1007/978-1-60761-688-7_6

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