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Strategies for immortalization of primary hepatocytes

  • Eva Ramboer
    Correspondence
    Corresponding author. Address: Department of Toxicology (FAFY), Center for Pharmaceutical Research (CePhaR), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. Tel.: +32 2 477 45 87; fax: +32 2 477 45 82.
    Affiliations
    Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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  • Bram De Craene
    Affiliations
    Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium

    Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
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  • Joery De Kock
    Affiliations
    Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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  • Tamara Vanhaecke
    Affiliations
    Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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  • Geert Berx
    Affiliations
    Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, 9052 Zwijnaarde, Belgium

    Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium
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  • Vera Rogiers
    Affiliations
    Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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  • Mathieu Vinken
    Affiliations
    Department of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussel, Belgium
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Open AccessPublished:June 07, 2014DOI:https://doi.org/10.1016/j.jhep.2014.05.046

      Summary

      The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications.

      Abbreviations:

      BAL (bio-artificial liver), (h)TERT ((human) telomerase reverse transcriptase), G (gap), DNA (deoxyribonucleic acid), mRNA (messenger ribonucleic acid), MAPK (mitogen-activated protein kinase), pRB (retinoblastoma protein), HGF (hepatocyte growth factor), EGF (epidermal growth factor), TGF (transforming growth factor), cdk(s) (cyclin-dependent kinase(s)), S (synthesis), M (mitosis), cdki (cdk inhibitor), PH (partial hepatectomy), SV40 Tag (simian virus 40 large T antigen), HPV16 (human papillomavirus type 16), Mo-MLV (Moloney Murine Leukemia virus), BMI-1 (B lymphoma Mo-MLV insertion region 1 homolog), HSV-TK (herpes simplex virus thymidine kinase), tTA (tetracycline transactivator), rtTA (reverse tetracycline transactivator), HIV (human immunodeficiency virus), HAC (human artificial chromosome), ALF (acute liver failure)

      Keywords

      Hepatocyte proliferation

      Priming phase and commitment to hepatocyte cell cycle progression

      Under normal conditions, the adult liver has very little proliferative activity. However, upon partial removal of liver tissue, the remaining intact hepatic lobes start to grow and liver mass is restored within seven to ten days due to the proliferation of mature hepatocytes [
      • Mangnall D.
      • Bird N.C.
      • Majeed A.W.
      The molecular physiology of liver regeneration following partial hepatectomy.
      ,
      • Malato Y.
      • Naqvi S.
      • Schürmann N.
      • Ng R.
      • Wang B.
      • Zape J.
      • et al.
      Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.
      ]. Multiple genes involved in cytokine networks become differentially expressed and regulate the initiation of this liver regeneration, a process called the “priming phase” [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Fausto N.
      • Campbell J.S.
      • Riehle K.J.
      Liver regeneration.
      ,
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ]. During this step, G0/G1 cell cycle transition and early G1 progression are accomplished and hepatocytes become responsive to mitogenic signals, which leads to deoxyribonucleic acid (DNA) replication [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ,
      • Fausto N.
      Liver regeneration.
      ]. During collagenase perfusion of the liver, a critical step in the isolation procedure of hepatocytes, the messenger ribonucleic acid (mRNA) levels of the proto-oncogenes c-Jun and c-Fos rapidly increase, suggesting that enzymatic liver dissociation triggers the G0/G1 cell cycle transition of hepatocytes [
      • Loyer P.
      • Cariou S.
      • Glaise D.
      • Bilodeau M.
      • Baffet G.
      • Guguen-Guillouzo C.
      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.
      ,
      • Etienne P.L.
      • Baffet G.
      • Desvergne B.
      • Boisnard-Rissel M.
      • Glaise D.
      • Guguen-Guillouzo C.
      Transient expression of c-fos and constant expression of c-myc in freshly isolated and cultured normal adult rat hepatocytes.
      ]. Indeed, collagenase perfusion of the liver, which is accompanied by release of the cytokine tumor necrosis factor α as well as activation of the intracellular nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinase (MAPK) pathways, can induce priming of quiescent hepatocytes [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Loyer P.
      • Cariou S.
      • Glaise D.
      • Bilodeau M.
      • Baffet G.
      • Guguen-Guillouzo C.
      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.
      ,
      • Paine A.J.
      • Andreakos E.
      Activation of signalling pathways during hepatocyte isolation: relevance to toxicology in vitro.
      ,
      • Liu M.L.
      • Mars W.M.
      • Zarnegar R.
      • Michalopoulos G.K.
      Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver.
      ,
      • Ilyin G.
      • Rescan C.
      • Rialland M.
      • Loyer P.
      • Baffet G.
      • Guguen-Guillouzo C.
      Growth control and cell cycle progression in cultured hepatocytes.
      ]. When the freshly isolated hepatocytes are plated, the sequentially increased expression of other proto-oncogenes, such as JunB, JunD, c-Myc, p53 and c-Ki-ras, indicates that the hepatocytes can proceed to the mid-late G1 phase [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Loyer P.
      • Cariou S.
      • Glaise D.
      • Bilodeau M.
      • Baffet G.
      • Guguen-Guillouzo C.
      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.
      ]. However, further progression towards the G1/S cell cycle boundary is only possible after stimulation with appropriate growth factors to overcome the mitogen-dependent mid-late G1 restriction point [
      • Loyer P.
      • Cariou S.
      • Glaise D.
      • Bilodeau M.
      • Baffet G.
      • Guguen-Guillouzo C.
      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.
      ]. This major checkpoint is regulated by the tumor suppressor retinoblastoma protein (pRB) and controls whether the cellular environment supports proliferation [
      • Mayhew C.N.
      • Bosco E.E.
      • Fox S.R.
      • Okaya T.
      • Tarapore P.
      • Schwemberger S.J.
      • et al.
      Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy.
      ,
      • Novák B.
      • Tyson J.J.
      A model for restriction point control of the mammalian cell cycle.
      ,
      • Schafer K.A.
      The cell cycle: a review.
      ]. The need for mitogenic signals to pursue cell cycling has also been shown in vivo, though intrinsic differences exist between the in vivo and in vitro conditions [
      • Fausto N.
      Liver regeneration.
      ,
      • Talarmin H.
      • Rescan C.
      • Cariou S.
      • Glaise D.
      • Zanninelli G.
      • Bilodeau M.
      • et al.
      The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes.
      ]. In vivo, normal adult hepatocytes return to the G0 state in the absence of growth factor stimulation, but that is not the case in vitro [
      • Mangnall D.
      • Bird N.C.
      • Majeed A.W.
      The molecular physiology of liver regeneration following partial hepatectomy.
      ,
      • Ilyin G.
      • Rescan C.
      • Rialland M.
      • Loyer P.
      • Baffet G.
      • Guguen-Guillouzo C.
      Growth control and cell cycle progression in cultured hepatocytes.
      ,
      • Talarmin H.
      • Rescan C.
      • Cariou S.
      • Glaise D.
      • Zanninelli G.
      • Bilodeau M.
      • et al.
      The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes.
      ]. After attaching to the culture dish, surviving cells remain at the mid-late G1 restriction point, do not proliferate and die early [
      • Ilyin G.
      • Rescan C.
      • Rialland M.
      • Loyer P.
      • Baffet G.
      • Guguen-Guillouzo C.
      Growth control and cell cycle progression in cultured hepatocytes.
      ,
      • Corlu A.
      • Ilyin G.
      • Cariou S.
      • Lamy I.
      • Loyer P.
      • Guguen-Guillouzo C.
      The coculture: a system for studying the regulation of liver differentiation/proliferation activity and its control.
      ].
      Several studies designated cyclin D1 as the major intracellular mediator of the mitogenic signals responsible for the regulation of hepatocyte proliferation [
      • Loyer P.
      • Cariou S.
      • Glaise D.
      • Bilodeau M.
      • Baffet G.
      • Guguen-Guillouzo C.
      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.
      ,
      • Talarmin H.
      • Rescan C.
      • Cariou S.
      • Glaise D.
      • Zanninelli G.
      • Bilodeau M.
      • et al.
      The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Nelsen C.J.
      • Rickheim D.G.
      • Timchenko N.A.
      • Stanley M.W.
      • Albrecht J.H.
      Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo.
      ,
      • Rickheim D.G.
      • Nelsen C.J.
      • Fassett J.T.
      • Timchenko N.A.
      • Hansen L.K.
      • Albrecht J.H.
      Differential regulation of cyclins D1 and D3 in hepatocyte proliferation.
      ,
      • Albrecht J.H.
      • Hansen L.K.
      Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.
      ]. As such, overexpression of D-type cyclins seems sufficient to overcome the mid-late G1 restriction point and trigger hepatocyte proliferation both, in vivo and in vitro, in the absence of mitogens [
      • Nelsen C.J.
      • Rickheim D.G.
      • Timchenko N.A.
      • Stanley M.W.
      • Albrecht J.H.
      Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo.
      ,
      • Albrecht J.H.
      • Hansen L.K.
      Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes.
      ,
      • Mullany L.K.
      • White P.
      • Hanse E.A.
      • Nelsen C.J.
      • Goggin M.M.
      • Mullany J.E.
      • et al.
      Distinct proliferative and transcriptional effects of the D-type cyclins in vivo.
      ]. Though, the latter has been challenged by Wierod et al. [
      • Wierød L.
      • Rosseland C.M.
      • Lindeman B.
      • Oksvold M.P.
      • Grøsvik H.
      • Skarpen E.
      • et al.
      CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes.
      ]. Interestingly, fetal hepatocytes, which express both cyclin D2 and D3, possess a high proliferation rate that is, at least partly, independent of mitogenic pathways and characterized by the constitutive phosphorylation of pRB [
      • Boylan J.M.
      • Gruppuso P.A.
      D-type cyclins and G1 progression during liver development in the rat.
      ,
      • Curran T.R.
      • Bahner R.I.
      • Oh W.
      • Gruppuso P.A.
      Mitogen-independent DNA synthesis by fetal rat hepatocytes in primary culture.
      ].
      Critical growth factors involved in hepatocyte cell cycling include hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF) α, heparin-binding EGF-like growth factor and amphiregulin [
      • Fausto N.
      • Campbell J.S.
      • Riehle K.J.
      Liver regeneration.
      ,
      • Riehle K.J.
      • Dan Y.Y.
      • Campbell J.S.
      • Fausto N.
      New concepts in liver regeneration.
      ]. Once past the mid-late G1 restriction point, hepatocytes are irreversibly committed to replicate and no longer require growth factors to complete the first cycle of cell proliferation [
      • Talarmin H.
      • Rescan C.
      • Cariou S.
      • Glaise D.
      • Zanninelli G.
      • Bilodeau M.
      • et al.
      The mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ]. From this point onwards, progression through the cell cycle proceeds autonomously and is driven by the sequential formation, activation and destruction of a series of structurally related serine/threonine protein kinase complexes, each composed of a regulatory and a catalytic subunit, cyclin and cyclin-dependent kinase (cdk), respectively [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ].

      Hepatocyte cell cycle regulation and control

      To date, at least 20 different cdk proteins and 30 cyclins have been identified in mammalian cells. However, only some are involved in cell cycle regulation [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ,
      • Malumbres M.
      • Harlow E.
      • Hunt T.
      • Hunter T.
      • Lahti J.M.
      • Manning G.
      • et al.
      Cyclin-dependent kinases: a family portrait.
      ]. Whereas the cdks are expressed throughout the hepatocyte cell cycle, with the notable exception of cdk1, most cyclins display a temporal expression profile, leading to periodic activation of their respective cdk counterparts [
      • Ilyin G.
      • Rescan C.
      • Rialland M.
      • Loyer P.
      • Baffet G.
      • Guguen-Guillouzo C.
      Growth control and cell cycle progression in cultured hepatocytes.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Guo Y.
      • Yang K.
      • Harwalkar J.
      • Nye J.M.
      • Mason D.R.
      • Garrett M.D.
      • et al.
      Phosphorylation of cyclin D1 at Thr 286 during S phase leads to its proteasomal degradation and allows efficient DNA synthesis.
      ]. Since these individual cyclin/cdk complexes perform unique functions in the cell cycle, their sequential assembly and activation dictates the order in which the cell cycle events occur [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ,
      • Chauhan A.
      • Lorenzen S.
      • Herzel H.
      • Bernard S.
      Regulation of mammalian cell cycle progression in the regenerating liver.
      ,
      • Vermeulen K.
      • Van Bockstaele D.R.
      • Berneman Z.N.
      The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.
      ] (Fig. 1). Nevertheless, subsequent progression through the S, G2 and M phases can be impeded by additional cell cycle checkpoints, which are switched on in response to unfavourable conditions [
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ]. In this context, checkpoints at the G1/S and G2/M boundaries ensure the orderly unfolding of different cell cycle events and inhibit cell cycling in response to DNA damage. Overall, mechanisms associated with activation of the p53/p21 pathway and suppression of the cdc25 family phosphatase activity are initiated, which results in reduced cdk activity and cell cycle arrest [
      • Novák B.
      • Tyson J.J.
      A model for restriction point control of the mammalian cell cycle.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Vermeulen K.
      • Van Bockstaele D.R.
      • Berneman Z.N.
      The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.
      ]. Indeed, in addition to cyclin binding, cdk activity is also regulated by a critical phosphorylation/dephosphorylation equilibrium and counteracted by cell cycle inhibitory proteins, called the cdk inhibitors (cdki) [
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ,
      • Vermeulen K.
      • Van Bockstaele D.R.
      • Berneman Z.N.
      The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.
      ] (Fig. 1). Based on their structure and the identity of their cdk targets, two families of cdki have been described, namely the Ink4 family and the Cip/Kip family. The former comprises four distinct proteins, namely p15, p16, p18, and p19, which are specific inhibitors of cdk4/6. The Cip/Kip family proteins, including p21, p27, and p57, bind and inhibit different cdk/cyclin complexes [
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Vermeulen K.
      • Van Bockstaele D.R.
      • Berneman Z.N.
      The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.
      ].
      Figure thumbnail gr1
      Fig. 1The hepatocyte cell cycle and its regulation. (A) The hepatocyte cell cycle, as in other eukaryotic cells, is composed of four phases namely the G1, S, G2, and M phase. Under physiological conditions, most hepatocytes in the adult liver escape the active cell cycle and enter a quiescent stage, known as the G0 phase. In this state, hepatocytes do not proliferate, but remain metabolically active. Upon appropriate stimulation, hepatocytes re-enter the cell cycle in the G1 phase [
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Cooper G.
      The eukaryotic cell cycle.
      ]. Progression through the mid-late G1 phase is growth factor-dependent. Once beyond the mitogen-dependent restriction point (RP), the cell cycle is completed autonomously, driven by the sequential activation of a series of structurally related serine/threonine protein kinases, the cyclin dependent kinases (cdk)
      [
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ]
      . In contrast with other mammalian cells, hepatocytes possess active cyclin A-cdk1 and cyclin B-cdk1 complexes during the S-phase of their cell cycle, which is suggested to allow rapid and efficient hepatocyte proliferation
      [
      • Garnier D.
      • Loyer P.
      • Ribault C.
      • Guguen-Guillouzo C.
      • Corlu A.
      Cyclin-dependent kinase 1 plays a critical role in DNA replication control during rat liver regeneration.
      ]
      . (B) The kinase activity of the cdks is tightly regulated by several different mechanisms, including binding to cyclins, binding by cdk inhibitors (cdki) and various phosphorylation/dephosphorylation events. For example full activation of the cyclin B-cdk1 complex requires its phosphorylation (P) on threonine 161 by the cdk-activating kinase (CAK). Other phosphorylation/dephosphorylation events fine-tune kinase activity and thereby facilitate proper mitotic action. The kinases Myt1 and Wee1 negatively affect kinase activity by phosphorylating cdk1 on threonine 14 (T14) and tyrosine 15 (Y15), whereas cdc25 phosphatase restores kinase activity by dephosphorylation of the same amino acids. Furthermore, Cip/Kip cdki can bind to the cyclin B/ckd1 complexes and inhibit their action [
      • Schafer K.A.
      The cell cycle: a review.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ,
      • Vermeulen K.
      • Van Bockstaele D.R.
      • Berneman Z.N.
      The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.
      ]. Adapted from [
      • Corlu A.
      • Loyer P.
      Regulation of the g1/s transition in hepatocytes: involvement of the cyclin-dependent kinase cdk1 in the DNA replication.
      ,
      • Albrecht J.H.
      • Mullany L.K.
      Cell cycle control in the liver.
      ,
      • Malumbres M.
      • Barbacid M.
      Mammalian cyclin-dependent kinases.
      ]. CAK, cdk-activating kinase; cdk(i), cyclin dependent kinase (inhibitor); G, gap; M, mitosis; P, phosphorylated; RP, restriction point; S, synthesis; T, threonine; Y, tyrosine.

      Hepatocyte senescence

      Following partial hepatectomy (PH), the remaining hepatocyte population needs to divide on average 1.6 times before the normal liver mass is restored and the regeneration is put back on hold [
      • Mangnall D.
      • Bird N.C.
      • Majeed A.W.
      The molecular physiology of liver regeneration following partial hepatectomy.
      ,
      • Fausto N.
      Liver regeneration.
      ,
      • Fausto N.
      • Campbell J.S.
      • Riehle K.J.
      Liver regeneration.
      ]. It has been suggested that TGFβ and activin A, known inhibitors of hepatocyte proliferation, as well as extracellular matrix-driven signals, underlie the termination of hepatocyte growth when the liver regeneration is completed [
      • Michalopoulos G.K.
      Liver regeneration.
      ,
      • Michalopoulos G.K.
      Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.
      ,
      • Oe S.
      • Lemmer E.R.
      • Conner E.A.
      • Factor V.M.
      • Levéen P.
      • Larsson J.
      • et al.
      Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice.
      ]. During chronic liver injury, human hepatocytes are repeatedly stimulated to proliferate due to iterative waves of liver destruction and regeneration [
      • Wiemann S.U.
      • Satyanarayana A.
      • Tsahuridu M.
      • Tillmann H.L.
      • Zender L.
      • Klempnauer J.
      • et al.
      Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis.
      ]. This in vivo proliferation capacity was further highlighted by the efficient repopulation of Fah−/−/Rag2−/−/Il2rg−/− mice with human adult hepatocytes for at least four sequential rounds [
      • Azuma H.
      • Paulk N.
      • Ranade A.
      • Dorrell C.
      • Al-Dhalimy M.
      • Ellis E.
      • et al.
      Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.
      ]. However, human hepatocytes cannot proliferate indefinitely. Liver cirrhosis is accompanied by a significant rate of hepatocellular senescence and characterized by considerable short hepatocyte telomeres [
      • Wiemann S.U.
      • Satyanarayana A.
      • Tsahuridu M.
      • Tillmann H.L.
      • Zender L.
      • Klempnauer J.
      • et al.
      Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis.
      ]. In humans, telomerase activity of most cell types is repressed early during development. Consequently, telomere DNA in proliferating somatic cells undergoes progressive attrition. Once a critical minimal length is reached, cellular growth is arrested irreversibly, a process known as replicative senescence, which was first described by Hayflick and Moorhead nearly 50 years ago [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Chiu C.P.
      • Harley C.B.
      Replicative senescence and cell immortality: the role of telomeres and telomerase.
      ,
      • Ozturk M.
      • Arslan-Ergul A.
      • Bagislar S.
      • Senturk S.
      • Yuzugullu H.
      Senescence and immortality in hepatocellular carcinoma.
      ,
      • Hayflick L.
      The limited in vitro lifetime of human diploid cell strains.
      ]. One way to overcome telomere-dependent senescence is by reactivating the telomerase activity with exogenous hTERT [
      • Kuilman T.
      • Michaloglou C.
      • Mooi W.J.
      • Peeper D.S.
      The essence of senescence.
      ,
      • Lee K.M.
      • Choi K.H.
      • Ouellette M.M.
      Use of exogenous hTERT to immortalize primary human cells.
      ,
      • Zhu J.
      • Wang H.
      • Bishop J.M.
      • Blackburn E.H.
      Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening.
      ]. In contrast to humans, rodents display substantial telomerase activity in several somatic tissues, including the liver [
      • Chiu C.P.
      • Harley C.B.
      Replicative senescence and cell immortality: the role of telomeres and telomerase.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ,
      • Gorbunova V.
      • Seluanov A.
      Coevolution of telomerase activity and body mass in mammals: from mice to beavers.
      ,
      • Nozawa K.
      • Kurumiya Y.
      • Yamamoto A.
      • Isobe Y.
      • Suzuki M.
      • Yoshida S.
      Up-regulation of telomerase in primary cultured rat hepatocytes.
      ,
      • Yamaguchi Y.
      • Nozawa K.
      • Savoysky E.
      • Hayakawa N.
      • Nimura Y.
      • Yoshida S.
      Change in telomerase activity of rat organs during growth and aging.
      ]. Their telomerase activity increases 24 h after PH and is enhanced by the preoperative treatment with EGF and HGF, but repressed by MAPK kinase inhibitors [
      • Inui T.
      • Shinomiya N.
      • Fukasawa M.
      • Kobayashi M.
      • Kuranaga N.
      • Ohkura S.
      • et al.
      Growth-related signaling regulates activation of telomerase in regenerating hepatocytes.
      ]. In primary rodent hepatocyte cultures, upregulation of telomerase activity was only notable or further enhanced after addition of growth factors to the culture medium [
      • Nozawa K.
      • Kurumiya Y.
      • Yamamoto A.
      • Isobe Y.
      • Suzuki M.
      • Yoshida S.
      Up-regulation of telomerase in primary cultured rat hepatocytes.
      ,
      • Inui T.
      • Shinomiya N.
      • Fukasawa M.
      • Kobayashi M.
      • Kuranaga N.
      • Ohkura S.
      • et al.
      Growth-related signaling regulates activation of telomerase in regenerating hepatocytes.
      ]. The high regeneration capacity, characteristic of rodent livers, may be linked to this strong telomerase activity [
      • Yamaguchi Y.
      • Nozawa K.
      • Savoysky E.
      • Hayakawa N.
      • Nimura Y.
      • Yoshida S.
      Change in telomerase activity of rat organs during growth and aging.
      ]. In this regard, serially transplanted adult mouse hepatocytes have been demonstrated to divide as many as 69 times [
      • Fausto N.
      Liver regeneration.
      ,
      • Overturf K.
      • al-Dhalimy M.
      • Ou C.N.
      • Finegold M.
      • Grompe M.
      Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.
      ].
      However, in vitro, both human and rat adult hepatocytes do not possess spontaneous cell growth and their proliferation capacity remains usually quite limited even when cultured under growth promoting conditions [
      • Fausto N.
      Liver regeneration.
      ,
      • Ilyin G.
      • Rescan C.
      • Rialland M.
      • Loyer P.
      • Baffet G.
      • Guguen-Guillouzo C.
      Growth control and cell cycle progression in cultured hepatocytes.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ,
      • Edwards A.
      • Michalopoulos G.
      Conditions for growth of hepatocytes in culture.
      ,
      • Block G.D.
      • Locker J.
      • Bowen W.C.
      • Petersen B.E.
      • Katyal S.
      • Strom S.C.
      • et al.
      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.
      ,
      • Runge D.M.
      • Runge D.
      • Dorko K.
      • Pisarov L.A.
      • Leckel K.
      • Kostrubsky V.E.
      • et al.
      Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.
      ]. The in vitro premature growth arrest, observed in primary hepatocyte cultures could be related to a telomere-independent senescence mechanism, which remains to be fully elucidated, but is suggested to involve tumour suppressor proteins and cdkis [
      • Ozturk M.
      • Arslan-Ergul A.
      • Bagislar S.
      • Senturk S.
      • Yuzugullu H.
      Senescence and immortality in hepatocellular carcinoma.
      ,
      • Ohtani N.
      • Yamakoshi K.
      • Takahashi A.
      • Hara E.
      The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression.
      ]. Indeed, several studies support the contribution of cdki p21 and/or p16 to the inhibition of DNA synthesis in primary hepatocyte cultures [
      • Auer K.L.
      • Park J.S.
      • Seth P.
      • Coffey R.J.
      • Darlington G.
      • Abo A.
      • et al.
      Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice.
      ,
      • Frémin C.
      • Bessard A.
      • Ezan F.
      • Gailhouste L.
      • Régeard M.
      • Le Seyec J.
      • et al.
      Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.
      ,
      • Harashima M.
      • Seki T.
      • Ariga T.
      • Niimi S.
      Role of p16(INK4a) in the inhibition of DNA synthesis stimulated by HGF or EGF in primary cultured rat hepatocytes.
      ,
      • Ilyin G.P.
      • Glaise D.
      • Gilot D.
      • Baffet G.
      • Guguen-Guillouzo C.
      Regulation and role of p21 and p27 cyclin-dependent kinase inhibitors during hepatocyte differentiation and growth.
      ,
      • Tombes R.M.
      • Auer K.L.
      • Mikkelsen R.
      • Valerie K.
      • Wymann M.P.
      • Marshall C.J.
      • et al.
      The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic.
      ]. In this respect, it was demonstrated that the second cell cycle G1 block caused by chronic MAPK pathway activation in mitogen stimulated primary hepatocyte cultures is partly related to p21 induction. Of note, transient MAPK pathway inhibition allows the establishment of multiple replication rounds in these hepatocyte cultures [
      • Frémin C.
      • Bessard A.
      • Ezan F.
      • Gailhouste L.
      • Régeard M.
      • Le Seyec J.
      • et al.
      Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.
      ].

      Hepatocyte immortalization strategies

      Immortalized hepatocytes are defined as a population of indefinitely dividing parenchymal cells that retain critical liver functions [
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ]. Since mature hepatocytes normally possess only limited growth potential when stimulated in vitro, immortalization strategies have been developed based mainly on the transduction or transfection of hepatocytes with well-known immortalizing genes. The most frequently used immortalization methods are (i) overexpression of viral oncogenes, (ii) forced expression of hTERT, or (iii) a combination of both [
      • Reid Y.
      • Gaddipati J.P.
      • Yadav D.
      • Kantor J.
      Establishment of a human neonatal hepatocyte cell line.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ]. Moreover, some other immortalization genes as well as conditional approaches for hepatocyte immortalization have been described (Fig. 2, Table 1, Table 2).
      Figure thumbnail gr2
      Fig. 2Hepatocyte immortalization strategies. Several hepatocyte immortalization strategies are available, including transduction or transfection of prototypical immortalization genes. Conditional immortalization by temperature-based regulation, recombinase-based control and transcriptional regulation have been introduced to establish growth-controlled cell lines. Adapted from
      [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ]
      . rtTA, reverse tetracycline transactivator; TRE, tetracycline responsive element; tTA, tetracycline transactivator.
      Table 1Overview of the functionality and immortalization strategy of in vitro established human and rodent hepatic cell lines.
      A1AGP, α1-acid glycoprotein; A1AT, α1-antitrypsin; ABC, ATP binding cassette; AFP, α-fetoprotein; AhR, aryl hydrocarbon receptor; ALB, albumin; ALF, acute liver failure; A2M, α2-macroglobulin; APO, apolipoprotein; Arnt, AhR nuclear translocator; ASGP(R), asialoglycoprotein (receptor); AST, aspartate aminotransferase; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog; CAR, constitutive androstane receptor; C/EBP, Ccaat-enhancer-binding protein; CD, cluster of differentiation; CK, cytokeratin; CLDN, claudin; CYP, cytochrome P450; DMSO, dimethyl sulphoxide; DPP, dipeptidyl peptidase; EH, epoxide hydrolase; EPCAM, epithelial cell adhesion molecule; GGT, γ-glutamyl transpeptidase; G6P, glucose-6-phosphate; GPX, glutathione peroxidase; GS, glutamine synthetase; GST, glutathione S-transferase; HBCF, human blood coagulation factor; HGFR, hepatocyte growth factor receptor; HNF, hepatocyte nuclear factor; HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase; IL, interleukin; INF, interferon; MDR, multidrug resistance protein; mRNA, messenger ribonucleic acid; MRP, multidrug resistance-associated protein; NADPH, nicotinamide adenine dinucleotide phosphate; NCAM, neural cell adhesion molecule; NH3, ammonia; PEPCK, phosphoenolpyruvate carboxykinase; PT, prothrombin; PXR, pregnane X receptor; Rb, retinoblastoma; SCID, severe combined immunodeficiency; SLC, solute carrier; SOD, superoxide dismutase; SV40 Tag, simian virus 40 large T antigen; TAT, tyrosine aminotransferase; TBL, total bilirubin; TF, transferrin; UGT, uridine diphosphate-glucuronosyltransferase.
      Table 2Overview of the available immortalization strategies.
      HSV-TK, herpes simplex virus thymidine kinase; hTERT, human telomerase reverse transcriptase; n.a., not applicable; SV40 Tag, simian virus 40 large T antigen.

      Immortalization genes

      Viral oncogenes

      Viral oncogenes include the adenoviral E1A/E1B genes, the simian virus 40 large T antigen (SV40 Tag) and the human papillomavirus 16 (HPV16) E6/E7 genes [
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ]. All of them have been used to establish hepatocyte-derived cell lines, such as C8-B, HepLL, HHL, AdPX3/4, Fa2N4, HepLi-4, and NKNT-3, suggesting that overexpression of viral oncogenes may be sufficient to overcome the premature in vitro growth arrest of cultured hepatocytes [
      • Clayton R.F.
      • Rinaldi A.
      • Kandyba E.E.
      • Edward M.
      • Willberg C.
      • Klenerman P.
      • et al.
      Liver cell lines for the study of hepatocyte functions and immunological response.
      ,
      • Woodworth C.D.
      • Isom H.C.
      Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA.
      ,
      • Li J.
      • Li L.J.
      • Cao H.C.
      • Sheng G.P.
      • Yu H.Y.
      • Xu W.
      • et al.
      Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy.
      ,
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Kobayashi N.
      • Fujiwara T.
      • Westerman K.A.
      • Inoue Y.
      • Sakaguchi M.
      • Noguchi H.
      • et al.
      Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.
      ,
      • Mills J.B.
      • Rose K.A.
      • Sadagopan N.
      • Sahi J.
      • de Morais S.M.
      Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line.
      ,
      • Zhao L.
      • Li J.
      • Lv G.
      • Zhang A.
      • Zhou P.
      • Yang Y.
      • et al.
      Evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs.
      ]. These viral oncogenes typically interfere with cell cycling by inhibiting the p16/pRB and p53 pathways [
      • Schafer K.A.
      The cell cycle: a review.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ]. Hepatic cell lines have also been developed from livers of transgenic rodents overexpressing the SV40 Tag [
      • Allen K.J.
      • Reyes R.
      • Demmler K.
      • Mercer J.F.
      • Williamson R.
      • Whitehead R.H.
      Conditionally immortalized mouse hepatocytes for use in liver gene therapy.
      ,
      • Bulera S.J.
      • Haas M.J.
      • Sattler C.A.
      • Li Y.
      • Pitot H.C.
      Cell lines with heterogeneous phenotypes result from a single isolation of albumin-sv40 T-antigen transgenic rat hepatocytes.
      ,
      • Yanai N.
      • Suzuki M.
      • Obinata M.
      Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.
      ].
      While the use of viral oncogenes, such as SV40 Tag, has been shown to be sufficient to immortalize rodent cells, overexpression of these oncogenes in human cells most likely only extends lifespan. Immortalization per se requires telomerase reactivation either through mutations or by the use of a second immortalizing gene, hTERT [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ,
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ,
      • McLean J.
      Immortalization strategies for mammalian cells.
      ,
      • Noguchi H.
      • Kobayashi N.
      Controlled expansion of mammalian cell populations by reversible immortalization.
      ]. Furthermore, the use of a combined strategy involving a viral oncogene and hTERT, has also been reported to produce more genetically stable cells [
      • Sinz M.
      • Kim S.
      Stem cells, immortalized cells and primary cells in ADMET assays.
      ,
      • Zhu J.
      • Wang H.
      • Bishop J.M.
      • Blackburn E.H.
      Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ,
      • Gabet A.S.
      • Accardi R.
      • Bellopede A.
      • Popp S.
      • Boukamp P.
      • Sylla B.S.
      • et al.
      Impairment of the telomere/telomerase system and genomic instability are associated with keratinocyte immortalization induced by the skin human papillomavirus type 38.
      ,
      • Kyo S.
      • Nakamura M.
      • Kiyono T.
      • Maida Y.
      • Kanaya T.
      • Tanaka M.
      • et al.
      Successful immortalization of endometrial glandular cells with normal structural and functional characteristics.
      ]. Indeed single use of viral oncogenes has often been demonstrated to induce chromosomal abnormalities [
      • Caporossi D.
      • Bacchetti S.
      Definition of adenovirus type 5 functions involved in the induction of chromosomal aberrations in human cells.
      ,
      • Schramayr S.
      • Caporossi D.
      • Mak I.
      • Jelinek T.
      • Bacchetti S.
      Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein.
      ,
      • Chang T.H.
      • Ray F.A.
      • Thompson D.A.
      • Schlegel R.
      Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells.
      ,
      • Toouli C.D.
      • Huschtscha L.I.
      • Neumann A.A.
      • Noble J.R.
      • Colgin L.M.
      • Hukku B.
      • et al.
      Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.
      ,
      • Stewart N.
      • Bacchetti S.
      Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells.
      ,
      • Ray F.A.
      • Peabody D.S.
      • Cooper J.L.
      • Cram L.S.
      • Kraemer P.M.
      SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.
      ,
      • Coursen J.D.
      • Bennett W.P.
      • Gollahon L.
      • Shay J.W.
      • Harris C.C.
      Genomic instability and telomerase activity in human bronchial epithelial cells during immortalization by human papillomavirus-16 E6 and E7 genes.
      ]. Even though karyotype analysis of newly produced hepatic cell lines has not routinely been performed, chromosomal abnormalities have been described in different cell lines even with combined immortalization [
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ,
      • Fukaya K.
      • Asahi S.
      • Nagamori S.
      • Sakaguchi M.
      • Gao C.
      • Miyazaki M.
      • et al.
      Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.
      ,
      • Kim B.H.
      • Sung S.R.
      • Choi E.H.
      • Kim Y.I.
      • Kim K.J.
      • Dong S.H.
      • et al.
      Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.
      ,
      • Smalley M.
      • Leiper K.
      • Tootle R.
      • McCloskey P.
      • O’Hare M.J.
      • Hodgson H.
      Immortalization of human hepatocytes by temperature-sensitive SV40 large-T antigen.
      ,
      • Pfeifer A.M.
      • Cole K.E.
      • Smoot D.T.
      • Weston A.
      • Groopman J.D.
      • Shields P.G.
      • et al.
      Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.
      ]. It is important to mention, however, that activation of an additional oncogene, such as Ras is usually needed to observe tumourigenicity [
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Woodworth C.D.
      • Kreider J.W.
      • Mengel L.
      • Miller T.
      • Meng Y.L.
      • Isom H.C.
      Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.
      ,
      • Guha C.
      • Chowdhury N.
      • Chowdhury J.
      Reversibly immortalized human hepatocytes: an eternal fountain of liver support?.
      ,
      • Isom H.C.
      • Woodworth C.D.
      • Meng Y.
      • Kreider J.
      • Miller T.
      • Mengel L.
      Introduction of the ras oncogene transforms a simian virus 40-immortalized hepatocyte cell line without loss of expression of albumin and other liver-specific genes.
      ].

      Human telomerase reverse transcriptase

      The single use of hTERT for immortalization has been suggested to avoid some of the genetic and phenotypic instabilities related to the use of oncogenes but is limited to a number of human cell types, including fetal and neonatal hepatocytes [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Chamuleau R.A.
      • Deurholt T.
      • Hoekstra R.
      Which are the right cells to be used in a bioartificial liver?.
      ,
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Reid Y.
      • Gaddipati J.P.
      • Yadav D.
      • Kantor J.
      Establishment of a human neonatal hepatocyte cell line.
      ,
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Waki K.
      • Anno K.
      • Ono T.
      • Ide T.
      • Chayama K.
      • Tahara H.
      Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development.
      ,
      • Wege H.
      • Chui M.S.
      • Le H.T.
      • Strom S.C.
      • Zern M.A.
      In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging.
      ]. Unlike adult hepatocytes, these immature cells can still proliferate in vitro and hence do not need cell cycle stimulation for immortalization [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Chamuleau R.A.
      • Deurholt T.
      • Hoekstra R.
      Which are the right cells to be used in a bioartificial liver?.
      ,
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Reid Y.
      • Gaddipati J.P.
      • Yadav D.
      • Kantor J.
      Establishment of a human neonatal hepatocyte cell line.
      ,
      • Curran T.R.
      • Bahner R.I.
      • Oh W.
      • Gruppuso P.A.
      Mitogen-independent DNA synthesis by fetal rat hepatocytes in primary culture.
      ,
      • Wege H.
      • Chui M.S.
      • Le H.T.
      • Strom S.C.
      • Zern M.A.
      In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging.
      ]. However, fetal and neonatal human hepatocytes do not possess indefinite growth potential because inactivation of telomerase causes replicative senescence. Consequently, they require overexpression of hTERT to become immortalized [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Reid Y.
      • Gaddipati J.P.
      • Yadav D.
      • Kantor J.
      Establishment of a human neonatal hepatocyte cell line.
      ,
      • Wege H.
      • Chui M.S.
      • Le H.T.
      • Strom S.C.
      • Zern M.A.
      In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging.
      ].
      Contradicting results have been reported when only hTERT was used for immortalization of human adult hepatocytes [
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ,
      • Okitsu T.
      • Kobayashi N.
      • Jun H.S.
      • Shin S.
      • Kim S.J.
      • Han J.
      • et al.
      Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.
      ,
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ]. As telomerase activity probably does not allow adult hepatocytes to overcome the proposed telomere-independent growth arrest, overexpression of hTERT may be insufficient to drive adult hepatocytes through the cell cycle [
      • Allen J.W.
      • Bhatia S.N.
      Improving the next generation of bioartificial liver devices.
      ,
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Lee K.M.
      • Choi K.H.
      • Ouellette M.M.
      Use of exogenous hTERT to immortalize primary human cells.
      ,
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ].

      Miscellaneous immortalization genes

      Specific combinations of immortalization genes, such as SV40 Tag with hTERT and B lymphoma Moloney Murine Leukemia virus (Mo-MLV) insertion region 1 homolog (Bmi-1), have been used to immortalize mature human hepatocytes. Bmi-1, like the viral oncogene HPV16E7, is involved in the inactivation of the p16/pRB pathway. On the other hand, simultaneous transduction with Bmi-1 and hTERT appears insufficient to immortalize the non-dividing hepatocytes [
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ]. Likewise, a combined HPV16E7/hTERT approach did not promote unlimited growth of human adult hepatocytes [
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ]. A particular cell line has been produced by co-transfection of human adult hepatocytes with p53 and pRB antisense constructs and plasmids that include E2F and cyclin D1 genes [
      • Werner A.
      • Duvar S.
      • Müthing J.
      • Büntemeyer H.
      • Kahmann U.
      • Lünsdorf H.
      • et al.
      Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system.
      ]. Furthermore, it seems that the hepatitis C core protein can also specifically immortalize mature human hepatocytes [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Ray R.B.
      • Meyer K.
      • Ray R.
      Hepatitis C virus core protein promotes immortalization of primary human hepatocytes.
      ,
      • Basu A.
      • Meyer K.
      • Ray R.B.
      • Ray R.
      Hepatitis C virus core protein is necessary for the maintenance of immortalized human hepatocytes.
      ]. This core protein is able to induce c-Myc and cyclin D1 expression in primary human hepatocytes via activation of the signal transducer and activator of transcription-3 pathway [
      • Basu A.
      • Meyer K.
      • Lai K.K.
      • Saito K.
      • Di Bisceglie A.M.
      • Grosso L.E.
      • et al.
      Microarray analyses and molecular profiling of Stat3 signaling pathway induced by hepatitis C virus core protein in human hepatocytes.
      ].
      In general, most of the generated hepatocyte-derived cell lines are not tumorigenic, but display reduced or only limited liver-specific functionality [
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ,
      • Kim B.H.
      • Sung S.R.
      • Choi E.H.
      • Kim Y.I.
      • Kim K.J.
      • Dong S.H.
      • et al.
      Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.
      ]. Taking into account that proliferation and differentiation are mutually exclusive in vitro, it has been shown that overexpression of the cdki p21 and the use of conditional immortalization strategies can stimulate to some extent differentiation of the cells [
      • Chamuleau R.A.
      • Deurholt T.
      • Hoekstra R.
      Which are the right cells to be used in a bioartificial liver?.
      ,
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ,
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Kobayashi N.
      • Fujiwara T.
      • Westerman K.A.
      • Inoue Y.
      • Sakaguchi M.
      • Noguchi H.
      • et al.
      Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.
      ,
      • Kim B.H.
      • Sung S.R.
      • Choi E.H.
      • Kim Y.I.
      • Kim K.J.
      • Dong S.H.
      • et al.
      Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.
      ,
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ,
      • Kunieda T.
      • Kobayashi N.
      • Sakaguchi M.
      • Okitsu T.
      • Totsugawa T.
      • Watanabe T.
      • et al.
      Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes.
      ,
      • Kobayashi N.
      • Kunieda T.
      • Sakaguchi M.
      • Okitsu T.
      • Totsugawa T.
      • Maruyama M.
      • et al.
      Active expression of p21 facilitates differentiation of immortalized human hepatocytes.
      ,
      • Fox I.J.
      • Chowdhury N.R.
      • Gupta S.
      • Kondapalli R.
      • Schilsky M.L.
      • Stockert R.J.
      • et al.
      Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.
      ,
      • Schumacher I.K.
      • Okamoto T.
      • Kim B.H.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • Fox I.J.
      Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy.
      ,
      • Zaret K.S.
      • DiPersio C.M.
      • Jackson D.A.
      • Montigny W.J.
      • Weinstat D.L.
      Conditional enhancement of liver-specific gene transcription.
      ]. Other anti-dedifferentiation strategies developed to counteract the loss of functionality in primary hepatocyte cultures, including co-culture systems and overexpression of liver-specific genes have also proven useful [
      • Watanabe T.
      • Shibata N.
      • Westerman K.A.
      • Okitsu T.
      • Allain J.E.
      • Sakaguchi M.
      • et al.
      Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers.
      ,
      • Inoue Y.
      • Miyazaki M.
      • Tsuji T.
      • Sakaguchi M.
      • Fukaya K.
      • Huh N.H.
      • et al.
      Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4alpha2 gene.
      ].

      Conditional immortalization strategies

      Conditional immortalization enables the development of growth-controlled cell lines. At least three strategies have been reported to conditionally immortalize hepatocytes, namely (i) temperature-based regulation, (ii) recombinase-based regulation and (iii) transcriptional regulation. All these methods rely on the observation that hepatocyte proliferation only takes place when immortalizing genes are expressed [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ] (Fig. 2, Table 1, Table 2).

      Temperature-based regulation

      This method uses a temperature-sensitive SV40 Tag mutant. The immortalizing gene is expressed and active only at the permissive temperature (33 °C), leading to the proliferation of hepatocytes. At higher temperatures (37–39 °C), the immortalization gene is inactivated and cell cycle progression is no longer stimulated [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ]. As no other temperature-labile immortalizing genes have yet been identified, this method is confined to SV40 Tag [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ]. Moreover, the use of this strategy is not accompanied by the excision of the immortalization gene from the genome and thus could present a potential risk of tumorigenesis [
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Guha C.
      • Chowdhury N.
      • Chowdhury J.
      Reversibly immortalized human hepatocytes: an eternal fountain of liver support?.
      ,
      • Paillard F.
      Reversible cell immortalization with the Cre-lox system.
      ]. Nevertheless, some conditionally immortalized hepatic cell lines are based on this principle, and these cell lines can be transplanted efficiently in rat models of acute liver failure and chronic hepatic encephalopathy, usually without occurrence of tumourigenicity [
      • Allen K.J.
      • Reyes R.
      • Demmler K.
      • Mercer J.F.
      • Williamson R.
      • Whitehead R.H.
      Conditionally immortalized mouse hepatocytes for use in liver gene therapy.
      ,
      • Yanai N.
      • Suzuki M.
      • Obinata M.
      Hepatocyte cell lines established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.
      ,
      • Kim B.H.
      • Sung S.R.
      • Choi E.H.
      • Kim Y.I.
      • Kim K.J.
      • Dong S.H.
      • et al.
      Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.
      ,
      • Fox I.J.
      • Chowdhury N.R.
      • Gupta S.
      • Kondapalli R.
      • Schilsky M.L.
      • Stockert R.J.
      • et al.
      Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.
      ,
      • Schumacher I.K.
      • Okamoto T.
      • Kim B.H.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • Fox I.J.
      Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy.
      ,
      • Zaret K.S.
      • DiPersio C.M.
      • Jackson D.A.
      • Montigny W.J.
      • Weinstat D.L.
      Conditional enhancement of liver-specific gene transcription.
      ,
      • Chen Y.
      • Li J.
      • Liu X.
      • Zhao W.
      • Wang Y.
      • Wang X.
      Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice.
      ,
      • Nakamura J.
      • Okamoto T.
      • Schumacher I.K.
      • Tabei I.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • et al.
      Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes.
      ]. However, concerns related to tumourigenicity form an important restriction to the clinical appreciation of immortalized human hepatocytes [
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ]. Importantly, the temperature shift associated with this methodology might induce changes in cellular properties, which can complicate the interpretation of the study outcome. A more sophisticated system, based on recombinase regulation, is thought to offer a solution for these issues [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Allen K.J.
      • Reyes R.
      • Demmler K.
      • Mercer J.F.
      • Williamson R.
      • Whitehead R.H.
      Conditionally immortalized mouse hepatocytes for use in liver gene therapy.
      ,
      • Fox I.J.
      • Chowdhury N.R.
      • Gupta S.
      • Kondapalli R.
      • Schilsky M.L.
      • Stockert R.J.
      • et al.
      Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.
      ,
      • Anastassiadis K.
      • Rostovskaya M.
      • Lubitz S.
      • Weidlich S.
      • Stewart A.F.
      Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.
      ,
      • May T.
      • Hauser H.
      • Wirth D.
      Transcriptional control of SV40 T-antigen expression allows a complete reversion of immortalization.
      ].

      Recombinase-based control

      The site-specific recombinase strategy uses recombinase expression to excise chromosomal DNA segments flanked by two recombination sequences and thereby irreversibly reverts immortalization [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Westerman K.A.
      • Leboulch P.
      Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.
      ]. Numerous site-specific recombination systems, including the Cre-loxP and the FLP-FRT system, have been used to establish reversible immortalization. These systems have different efficiencies, whereby the Cre-loxP system stands out [
      • Paillard F.
      Reversible cell immortalization with the Cre-lox system.
      ,
      • Westerman K.A.
      • Leboulch P.
      Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.
      ]. In this system, immortalization genes are flanked by two identical DNA sequences, called LoxP sites. The excision of these genes is regulated by Cre recombinase [
      • Cascio S.M.
      Novel strategies for immortalization of human hepatocytes.
      ,
      • Paillard F.
      Reversible cell immortalization with the Cre-lox system.
      ]. Proper reversion thus depends on the efficient transfer of the recombinase gene [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ]. More recently, a new method based on tamoxifen-mediated self-excision has been established, rendering secondary virus-mediated transfer of the recombinase gene superfluous [
      • Zhao L.
      • Li J.
      • Lv G.
      • Zhang A.
      • Zhou P.
      • Yang Y.
      • et al.
      Evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs.
      ,
      • Okitsu T.
      • Kobayashi N.
      • Jun H.S.
      • Shin S.
      • Kim S.J.
      • Han J.
      • et al.
      Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.
      ,
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ]. Furthermore, the suicide gene herpes simplex virus thymidine kinase (HSV-TK) has been introduced in the recombination construct as negative selection marker. Using this strategy, cells that still express the immortalization gene and HSV-TK gene, due to improper recombination, can be eliminated by exposure to ganciclovir [
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ,
      • Paillard F.
      Reversible cell immortalization with the Cre-lox system.
      ]. Reversible immortalization of numerous hepatocyte-derived cell lines, including C8-B, NKNT-3, IHH, and 16T-3 depends on this recombinase-based control approach [
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ,
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Kobayashi N.
      • Fujiwara T.
      • Westerman K.A.
      • Inoue Y.
      • Sakaguchi M.
      • Noguchi H.
      • et al.
      Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.
      ,
      • Okitsu T.
      • Kobayashi N.
      • Jun H.S.
      • Shin S.
      • Kim S.J.
      • Han J.
      • et al.
      Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.
      ,
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ,
      • Ito M.
      • Ito R.
      • Yoshihara D.
      • Ikeno M.
      • Kamiya M.
      • Suzuki N.
      • et al.
      Immortalized hepatocytes using human artificial chromosome.
      ,
      • Kobayashi N.
      • Noguchi H.
      • Fujiwara T.
      • Tanaka N.
      Establishment of a reversibly immortalized human hepatocyte cell line by using Cre/loxP site-specific recombination.
      ].

      Transcriptional regulation

      In this method, immortalization reversibility is obtained by transcriptional control of immortalization gene expression and not by recombinase activity. In this way, the risk of chromosomal rearrangement is avoided and repeated cycles of hepatocyte proliferation and growth arrest are allowed [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ,
      • Anastassiadis K.
      • Rostovskaya M.
      • Lubitz S.
      • Weidlich S.
      • Stewart A.F.
      Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.
      ,
      • May T.
      • Hauser H.
      • Wirth D.
      Transcriptional control of SV40 T-antigen expression allows a complete reversion of immortalization.
      ]. Transcription of immortalizing genes can be controlled by using an artificial promoter/transactivator system, such as the well-known tetracycline system [
      • Lipps C.
      • May T.
      • Hauser H.
      • Wirth D.
      Eternity and functionality – rational access to physiologically relevant cell lines.
      ]. Two approaches are currently available, the tet-off and the tet-on system, which are composed of a tetracycline-regulated promoter and a tetracycline transactivator (tTA) or reverse tetracycline transactivator (rtTA), respectively. When doxycycline is added to the cell culture medium, it binds to the transactivator. In the tet-on systems, bound rtTA interacts with the tetracycline-regulated promoter and induces the expression of the regulated gene. When using the tet-off method, immortalization genes are expressed in the absence of doxycycline, since only unbound tTA can interact with the gene promoter [
      • Anastassiadis K.
      • Rostovskaya M.
      • Lubitz S.
      • Weidlich S.
      • Stewart A.F.
      Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.
      ,
      • Jazwa A.
      • Florczyk U.
      • Jozkowicz A.
      • Dulak J.
      Gene therapy on demand: site specific regulation of gene therapy.
      ]. The tet-on approach has been successfully used to produce a fetal liver cell line [
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ]. A drawback of this method, however, is the possible leaky transgene expression caused by undesired rtTA-tetracycline promoter binding in the absence of doxycycline [
      • Anastassiadis K.
      • Rostovskaya M.
      • Lubitz S.
      • Weidlich S.
      • Stewart A.F.
      Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.
      ,
      • Jazwa A.
      • Florczyk U.
      • Jozkowicz A.
      • Dulak J.
      Gene therapy on demand: site specific regulation of gene therapy.
      ]. A tighter regulation of the transgene expression can be obtained by combining the rtTA system with a tetracycline-controlled transcriptional silencer [
      • Jazwa A.
      • Florczyk U.
      • Jozkowicz A.
      • Dulak J.
      Gene therapy on demand: site specific regulation of gene therapy.
      ].

      Gene transfer

      An effective gene transfer method is of utmost importance for immortalizing hepatocytes [
      • McLean J.
      Immortalization strategies for mammalian cells.
      ]. Different non-viral and viral methods have been used to generate immortalized hepatocyte-derived cell lines, namely plasmid transfection, viral transduction and the use of human artificial chromosomes (Table 2).

      Plasmid transfection

      Various approaches are available for transfecting plasmids into primary hepatocytes [
      • McLean J.
      Immortalization strategies for mammalian cells.
      ,
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ]. Due to immortalization, stably transfected cells are selected, allowing simple transfection procedures to be used [
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ]. Examples of common transfection methods that have been used to immortalize hepatocytes include calcium phosphate precipitation and electroporation [
      • Watanabe N.
      • Odagiri H.
      • Totsuka E.
      • Sasaki M.
      A new method to immortalize primary cultured rat hepatocytes.
      ,
      • Woodworth C.D.
      • Isom H.C.
      Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA.
      ,
      • Macdonald C.
      • Willett B.
      The immortalisation of rat hepatocytes by transfection with SV40 sequences.
      ,
      • Woodworth C.
      • Secott T.
      • Isom H.C.
      Transformation of rat hepatocytes by transfection with simian virus 40 DNA to yield proliferating differentiated cells.
      ]. However, both approaches typically display low gene transfer efficiencies and high hepatocyte toxicity [
      • McLean J.
      Immortalization strategies for mammalian cells.
      ,
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ]. Replacement of calcium by strontium eliminates toxicity but the gene transfer efficiency remains low [
      • McLean J.
      Immortalization strategies for mammalian cells.
      ]. Other researchers explored liposomes as gene carriers for hepatocyte immortalization [
      • Fukaya K.
      • Asahi S.
      • Nagamori S.
      • Sakaguchi M.
      • Gao C.
      • Miyazaki M.
      • et al.
      Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.
      ,
      • Li J.
      • Li L.J.
      • Cao H.C.
      • Sheng G.P.
      • Yu H.Y.
      • Xu W.
      • et al.
      Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy.
      ,
      • Werner A.
      • Duvar S.
      • Müthing J.
      • Büntemeyer H.
      • Kahmann U.
      • Lünsdorf H.
      • et al.
      Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system.
      ,
      • Schippers I.J.
      • Moshage H.
      • Roelofsen H.
      • Müller M.
      • Heymans H.S.
      • Ruiters M.
      • et al.
      Immortalized human hepatocytes as a tool for the study of hepatocytic (de-) differentiation.
      ,
      • Kobayashi N.
      • Noguchi H.
      • Watanabe T.
      • Matsumura T.
      • Totsugawa T.
      • Fujiwara T.
      • et al.
      Role of immortalized hepatocyte transplantation in acute liver failure.
      ,
      • Noguchi M.
      • Hirohashi S.
      Cell lines from non-neoplastic liver and hepatocellular carcinoma tissue from a single patient.
      ]. When properly optimized, lipid-mediated gene transfer can achieve high gene transfer efficiencies compared to other transfection approaches [
      • McLean J.
      Immortalization strategies for mammalian cells.
      ]. Furthermore, using hepatocyte-specific ligands, more hepatocyte-specific transfections can be achieved [
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ].

      Viral transduction

      Transduction with viral particles covers a widely used methodology for gene transfer. Among the available viral vectors, retroviral and lentiviral vectors induce stable integration of the immortalization gene and thus generate sustained transgene expression in the progeny [
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ,
      • Kazuki Y.
      • Oshimura M.
      Human artificial chromosomes for gene delivery and the development of animal models.
      ]. Furthermore, these vectors do not provoke harmful immune responses and allow integration of large genes [
      • Zahler M.H.
      • Irani A.
      • Malhi H.
      • Reutens A.T.
      • Albanese C.
      • Bouzahzah B.
      • et al.
      The application of a lentiviral vector for gene transfer in fetal human hepatocytes.
      ]. Retroviral vectors, such as the Mo-MLV-derived vectors, have been frequently used to establish human and rodent hepatic cell lines [
      • Wege H.
      • Le H.T.
      • Chui M.S.
      • Liu L.
      • Wu J.
      • Giri R.
      • et al.
      Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.
      ,
      • Reid Y.
      • Gaddipati J.P.
      • Yadav D.
      • Kantor J.
      Establishment of a human neonatal hepatocyte cell line.
      ,
      • Clayton R.F.
      • Rinaldi A.
      • Kandyba E.E.
      • Edward M.
      • Willberg C.
      • Klenerman P.
      • et al.
      Liver cell lines for the study of hepatocyte functions and immunological response.
      ,
      • Cai J.
      • Ito M.
      • Westerman K.A.
      • Kobayashi N.
      • Leboulch P.
      • Fox I.J.
      Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.
      ,
      • Zhao L.
      • Li J.
      • Lv G.
      • Zhang A.
      • Zhou P.
      • Yang Y.
      • et al.
      Evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs.
      ,
      • Kim B.H.
      • Sung S.R.
      • Choi E.H.
      • Kim Y.I.
      • Kim K.J.
      • Dong S.H.
      • et al.
      Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.
      ,
      • Smalley M.
      • Leiper K.
      • Tootle R.
      • McCloskey P.
      • O’Hare M.J.
      • Hodgson H.
      Immortalization of human hepatocytes by temperature-sensitive SV40 large-T antigen.
      ,
      • Pfeifer A.M.
      • Cole K.E.
      • Smoot D.T.
      • Weston A.
      • Groopman J.D.
      • Shields P.G.
      • et al.
      Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.
      ,
      • Waki K.
      • Anno K.
      • Ono T.
      • Ide T.
      • Chayama K.
      • Tahara H.
      Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development.
      ,
      • Okitsu T.
      • Kobayashi N.
      • Jun H.S.
      • Shin S.
      • Kim S.J.
      • Han J.
      • et al.
      Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.
      ,
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ,
      • Fox I.J.
      • Chowdhury N.R.
      • Gupta S.
      • Kondapalli R.
      • Schilsky M.L.
      • Stockert R.J.
      • et al.
      Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.
      ,
      • Schumacher I.K.
      • Okamoto T.
      • Kim B.H.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • Fox I.J.
      Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy.
      ,
      • Chen Y.
      • Li J.
      • Liu X.
      • Zhao W.
      • Wang Y.
      • Wang X.
      Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice.
      ,
      • Kobayashi N.
      • Noguchi H.
      • Fujiwara T.
      • Westerman K.A.
      • Leboulch P.
      • Tanaka N.
      Establishment of a highly differentiated immortalized adult human hepatocyte cell line by retroviral gene transfer.
      ,
      • Pan X.
      • Li J.
      • Du W.
      • Yu X.
      • Zhu C.
      • Yu C.
      • et al.
      Establishment and characterization of immortalized human hepatocyte cell line for applications in bioartificial livers.
      ]. A major flaw in this system is its inability to transduce non-dividing cells, which makes it unsuitable for non-proliferating cells, including hepatocytes [
      • Zahler M.H.
      • Irani A.
      • Malhi H.
      • Reutens A.T.
      • Albanese C.
      • Bouzahzah B.
      • et al.
      The application of a lentiviral vector for gene transfer in fetal human hepatocytes.
      ,
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ]. Even when growth factors are added to the cell culture medium to induce hepatocyte mitosis, the efficiency of transduction often remains limited [
      • Wang X.
      • Mani P.
      • Sarkar D.P.
      • Roy-Chowdhury N.
      • Roy-Chowdhury J.
      Ex vivo gene transfer into hepatocytes.
      ,
      • Zahler M.H.
      • Irani A.
      • Malhi H.
      • Reutens A.T.
      • Albanese C.
      • Bouzahzah B.
      • et al.
      The application of a lentiviral vector for gene transfer in fetal human hepatocytes.
      ,
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ,
      • Ohashi K.
      • Park F.
      • Kay M.A.
      Hepatocyte transplantation: clinical and experimental application.
      ]. Lentiviral vectors derived from the human immunodeficiency virus (HIV) can tackle these issues and transduce both dividing and non-dividing cells by using virus at a relatively high titer [
      • Zahler M.H.
      • Irani A.
      • Malhi H.
      • Reutens A.T.
      • Albanese C.
      • Bouzahzah B.
      • et al.
      The application of a lentiviral vector for gene transfer in fetal human hepatocytes.
      ,
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ,
      • Ohashi K.
      • Park F.
      • Kay M.A.
      Hepatocyte transplantation: clinical and experimental application.
      ,
      • Selden C.
      • Mellor N.
      • Rees M.
      • Laurson J.
      • Kirwan M.
      • Escors D.
      • et al.
      Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes.
      ]. Moreover, lentiviral vectors can provide high transduction efficiencies without affecting the differentiated hepatic phenotype [
      • Zahler M.H.
      • Irani A.
      • Malhi H.
      • Reutens A.T.
      • Albanese C.
      • Bouzahzah B.
      • et al.
      The application of a lentiviral vector for gene transfer in fetal human hepatocytes.
      ,
      • Ohashi K.
      • Park F.
      • Kay M.A.
      Hepatocyte transplantation: clinical and experimental application.
      ,
      • Zamule S.M.
      • Strom S.C.
      • Omiecinski C.J.
      Preservation of hepatic phenotype in lentiviral-transduced primary human hepatocytes.
      ]. Although lentiviral vectors lack hepatocyte specificity, the use of hepatocyte specific promoters can restrict the expression of lentiviral genes to the parenchymal liver cells [
      • Selden C.
      • Mellor N.
      • Rees M.
      • Laurson J.
      • Kirwan M.
      • Escors D.
      • et al.
      Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes.
      ]. Several studies have demonstrated appropriate gene transfer for immortalization of human adult and fetal hepatocytes [
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai H.
      • Matsumoto S.
      • et al.
      Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy.
      ,
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ]. Rodent hepatocytes, especially murine hepatocytes are considerably resistant to HIV vector-mediated transduction. This resistance has been related to a block in the immediate-early phase of infection [
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ]. In addition to the use of higher viral titers, cell culture medium supplied with growth factors, namely EGF and to a lesser extent HGF, was found to improve lentiviral transduction efficacy of primary mouse hepatocytes [
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ,
      • Rothe M.
      • Rittelmeyer I.
      • Iken M.
      • Rüdrich U.
      • Schambach A.
      • Glage S.
      • et al.
      Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes.
      ]. Similarly, when transducing human adult and fetal hepatocytes, the use of growth factors markedly upregulated the expression of lentiviral genes. Consequently, this transduction approach offers the possibility to reduce the viral load, which as such lowers cost and reduces cellular toxicity [
      • Selden C.
      • Mellor N.
      • Rees M.
      • Laurson J.
      • Kirwan M.
      • Escors D.
      • et al.
      Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes.
      ]. Also the antioxidant, vitamin E proved to significantly enhance lentiviral transduction rates of human and rat adult hepatocytes [
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ].

      Human artificial chromosomes

      The generation of a particular rat hepatic cell line was made possible by a more recent gene transfer method, namely through generation of a human artificial chromosome (HAC) [
      • Ito M.
      • Ito R.
      • Yoshihara D.
      • Ikeno M.
      • Kamiya M.
      • Suzuki N.
      • et al.
      Immortalized hepatocytes using human artificial chromosome.
      ,
      • Ito M.
      • Ikeno M.
      • Nagata H.
      • Yamamoto T.
      • Hiroguchi A.
      • Fox I.J.
      • et al.
      Treatment of nonalbumin rats by transplantation of immortalized hepatocytes using artificial human chromosome.
      ]. Although this method generally has lower transfer efficiency than the use of viral vectors, the HACs possess many properties of the ideal gene delivery vector. These include mitotically stable episomal maintenance and incorporation of large genes under control of their regulatory elements, allowing a correct, physiologically regulated transgene expression. Furthermore, due to their episomal nature, integration-related complications, such as oncogenesis, should be avoided [
      • Kazuki Y.
      • Oshimura M.
      Human artificial chromosomes for gene delivery and the development of animal models.
      ]. Immortalization of human fibroblasts using HAC-mediated episomal expression of hTERT has also been described, potentially offering new perspectives for hepatocyte immortalization [
      • Shitara S.
      • Kakeda M.
      • Nagata K.
      • Hiratsuka M.
      • Sano A.
      • Osawa K.
      • et al.
      Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector.
      ].

      Application of immortalized hepatic cell lines

      It has repeatedly been postulated that immortalized hepatic cell lines, which could offer an unlimited supply of well-characterized, pathogen-free cells, may represent an attractive alternative for primary hepatocytes in several clinical applications as well as fundamental and applied research [
      • Guha C.
      • Chowdhury N.
      • Chowdhury J.
      Reversibly immortalized human hepatocytes: an eternal fountain of liver support?.
      ,
      • Ito M.
      • Ikeno M.
      • Nagata H.
      • Yamamoto T.
      • Hiroguchi A.
      • Fox I.J.
      • et al.
      Treatment of nonalbumin rats by transplantation of immortalized hepatocytes using artificial human chromosome.
      ,
      • Cai J.
      • Ito M.
      • Nagata H.
      • Westerman K.A.
      • Lafleur D.
      • Chowdhury J.R.
      • et al.
      Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes.
      ]. So far, multiple studies based on immortalized hepatocytes have already been performed.

      Clinical application

      Hepatocyte transplantation

      The use of different animal models of hepatic impairment made it possible to demonstrate the therapeutic efficiency of transplanted cell lines. In this regard, it was shown that transplantation of conditional immortalized rat hepatocytes could protect portacaval-shunted rats from hyperammonemia-induced hepatic encephalopathy [
      • Schumacher I.K.
      • Okamoto T.
      • Kim B.H.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • Fox I.J.
      Transplantation of conditionally immortalized hepatocytes to treat hepatic encephalopathy.
      ,
      • Cai J.
      • Ito M.
      • Nagata H.
      • Westerman K.A.
      • Lafleur D.
      • Chowdhury J.R.
      • et al.
      Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes.
      ], improve survival of rats with acute liver failure (ALF) [
      • Nakamura J.
      • Okamoto T.
      • Schumacher I.K.
      • Tabei I.
      • Chowdhury N.R.
      • Chowdhury J.R.
      • et al.
      Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes.
      ], adjust for bilirubin conjugation defect in Gunn rats [
      • Tada K.
      • Roy-Chowdhury N.
      • Prasad V.
      • Kim B.H.
      • Manchikalapudi P.
      • Fox I.J.
      • et al.
      Long-term amelioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes.
      ,
      • Kim B.H.
      • Han Y.S.
      • Dong S.H.
      • Kim H.J.
      • Chang Y.W.
      • Lee J.I.
      • et al.
      Temporary amelioration of bilirubin conjugation defect in Gunn rats by transplanting conditionally immortalized hepatocytes.
      ], and correct the global hepatic abnormalities associated with end-stage liver failure in cirrhotic animals [
      • Cai J.
      • Ito M.
      • Nagata H.
      • Westerman K.A.
      • Lafleur D.
      • Chowdhury J.R.
      • et al.
      Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes.
      ]. Likewise, several human adult and fetal hepatic cell lines, including HHE6E7T1, NKNT-3, IHH, HepCL, 16T-3, and OUMS-29 were confirmed to promote survival in a pig [
      • Totsugawa T.
      • Yong C.
      • Rivas-Carrillo J.D.
      • Soto-Gutierrez A.
      • Navarro-Alvarez N.
      • Noguchi H.
      • et al.
      Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination.
      ], rat [
      • Kobayashi N.
      • Noguchi H.
      • Fujiwara T.
      • Tanaka N.
      Xenotransplantation of immortalized human hepatocytes for experimental acute liver failure in rats.
      ] or mice [
      • Nguyen T.H.
      • Mai G.
      • Villiger P.
      • Oberholzer J.
      • Salmon P.
      • Morel P.
      • et al.
      Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes.
      ,
      • Chen Y.
      • Li J.
      • Liu X.
      • Zhao W.
      • Wang Y.
      • Wang X.
      Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice.
      ,
      • Tsuruga Y.
      • Kiyono T.
      • Matsushita M.
      • Takahashi T.
      • Kasai N.
      • Matsumoto S.
      • et al.
      Effect of intrasplenic transplantation of immortalized human hepatocytes in the treatment of acetaminophen-induced acute liver failure SCID mice.
      ] model of ALF. Furthermore, YOCK-13, an insulin-producing human hepatic cell line was reported to control diabetes when transplanted into totally pancreatectomized diabetic pigs [
      • Okitsu T.
      • Kobayashi N.
      • Jun H.S.
      • Shin S.
      • Kim S.J.
      • Han J.
      • et al.
      Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs.
      ].

      Bioartificial liver systems

      For large-scale applications that rely on in vitro hepatic functionality, such as BAL systems, the development of a hepatic cell line that combines both in vitro hepatic function and proliferation capacity would be of great value.
      Two human fetal hepatic cell lines, namely HepLi-4 and cBAL111, have already been evaluated as a potential cell source for BAL systems [
      • Zhao L.
      • Li J.
      • Lv G.
      • Zhang A.
      • Zhou P.
      • Yang Y.
      • et al.
      Evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs.
      ,
      • Poyck P.P.
      • van Wijk A.C.
      • van der Hoeven T.V.
      • de Waart D.R.
      • Chamuleau R.A.
      • van Gulik T.M.
      • et al.
      Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.
      ]. However, it was revealed that both cell lines possessed insufficient hepatic functionality to be applicable for in vitro applications. The need for in vitro culture conditions that mimic the in vivo situation and promote hepatocyte differentiation in vitro was clearly emphasized [
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ,
      • Zhao L.
      • Li J.
      • Lv G.
      • Zhang A.
      • Zhou P.
      • Yang Y.
      • et al.
      Evaluation of a reversibly immortalized human hepatocyte line in bioartificial liver in pigs.
      ,
      • Poyck P.P.
      • van Wijk A.C.
      • van der Hoeven T.V.
      • de Waart D.R.
      • Chamuleau R.A.
      • van Gulik T.M.
      • et al.
      Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.
      ]. This was further supported by experiments, which showed that cBAL111 cells are able to partly differentiate into functional hepatocytes once transplanted in vivo [
      • Deurholt T.
      • van Til N.P.
      • Chhatta A.A.
      • ten Bloemendaal L.
      • Schwartlander R.
      • Payne C.
      • et al.
      Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.
      ].
      Different human adult hepatic cell lines have also been proposed as possible candidates for BAL application, but as for the modified fetal hepatic cell line, OUMS-29/H-11, data on efficacy in animal models of severe liver failure are currently lacking [
      • Werner A.
      • Duvar S.
      • Müthing J.
      • Büntemeyer H.
      • Kahmann U.
      • Lünsdorf H.
      • et al.
      Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system.
      ,
      • Pan X.
      • Li J.
      • Du W.
      • Yu X.
      • Zhu C.
      • Yu C.
      • et al.
      Establishment and characterization of immortalized human hepatocyte cell line for applications in bioartificial livers.
      ,
      • Werner A.
      • Duvar S.
      • Müthing J.
      • Büntemeyer H.
      • Lünsdorf H.
      • Strauss M.
      • et al.
      Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers.
      ,
      • Yu C.B.
      • Lv G.L.
      • Pan X.P.
      • Chen Y.S.
      • Cao H.C.
      • Zhang Y.M.
      • et al.
      In vitro large-scale cultivation and evaluation of microencapsulated immortalized human hepatocytes (HepLL) in roller bottles.
      ,
      • Akiyama I.
      • Tomiyama K.
      • Sakaguchi M.
      • Takaishi M.
      • Mori M.
      • Hosokawa M.
      • et al.
      Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor.
      ,
      • Nibourg G.A.
      • Chamuleau R.A.
      • van Gulik T.M.
      • Hoekstra R.
      Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.
      ]. However, the production of ammonia [
      • Werner A.
      • Duvar S.
      • Müthing J.
      • Büntemeyer H.
      • Lünsdorf H.
      • Strauss M.
      • et al.
      Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers.
      ] or possible inability to eliminate ammonia [
      • Pan X.
      • Li J.
      • Du W.
      • Yu X.
      • Zhu C.
      • Yu C.
      • et al.
      Establishment and characterization of immortalized human hepatocyte cell line for applications in bioartificial livers.
      ] are undesirable features for a BAL system [
      • Nibourg G.A.
      • Chamuleau R.A.
      • van Gulik T.M.
      • Hoekstra R.
      Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.
      ].
      Another modified adult hepatic cell line, composed of TTNT cells overexpressing IL-1 Ra, has already been tested and was not able to improve survival of an ALF rat model [
      • Nibourg G.A.
      • Chamuleau R.A.
      • van Gulik T.M.
      • Hoekstra R.
      Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.
      ].

      Fundamental and applied research

      Nowadays, human and rodent hepatic cell lines, such as CWSV [
      • Buzzelli M.D.
      • Nagarajan M.
      • Radtka J.F.
      • Shumate M.L.
      • Navaratnarajah M.
      • Lang C.H.
      • et al.
      Nuclear factor-kappaB mediates the inhibitory effects of tumor necrosis factor-alpha on growth hormone-inducible gene expression in liver.
      ,
      • Ahmed T.A.
      • Buzzelli M.D.
      • Lang C.H.
      • Capen J.B.
      • Shumate M.L.
      • Navaratnarajah M.
      • et al.
      Interleukin-6 inhibits growth hormone-mediated gene expression in hepatocytes.
      ], H2.35 [
      • Bai J.
      • Li J.
      • Mao Q.
      Construction of a single lentiviral vector containing tetracycline-inducible Alb-uPA for transduction of uPA expression in murine hepatocytes.
      ,
      • Samavati L.
      • Lee I.
      • Mathes I.
      • Lottspeich F.
      • Hüttemann M.
      Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase.
      ], NeHepLxHT [
      • Gao Y.
      • Theng S.S.
      • Zhuo J.
      • Teo W.B.
      • Ren J.
      • Lee C.G.
      FAT10, an ubiquitin-like protein, confers malignant properties in non-tumorigenic and tumorigenic cells.
      ], OUMS-29 [
      • Tomimaru Y.
      • Xu C.Q.
      • Nambotin S.B.
      • Yan T.
      • Wands J.R.
      • Kim M.
      Loss of exon 4 in a human T-cell factor-4 isoform promotes hepatic tumourigenicity.
      ], and THLE [
      • Rand A.A.
      • Rooney J.P.
      • Butt C.M.
      • Meyer J.N.
      • Mabury S.A.
      Cellular toxicity associated with exposure to perfluorinated carboxylates (PFCAs) and their metabolic precursors.
      ,
      • Krajka-Kuźniak V.
      • Paluszczak J.
      • Baer-Dubowska W.
      Xanthohumol induces phase II enzymes via Nrf2 in human hepatocytes in vitro.
      ] are still being used for fundamental research. In this regard, a lot of investigations related to hepatotropic viruses have been performed on TPH1 cells [
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Dash S.
      • Kanda T.
      • Ray R.
      • et al.
      Hepatitis C virus infection impairs IRF-7 translocation and Alpha interferon synthesis in immortalized human hepatocytes.
      ,
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Kim H.
      • Ray R.
      • Ray R.B.
      ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
      ]. Furthermore, a murine model of HBV viremia, based on immortalized human hepatocytes transfected with hepatitis B virus DNA, has been created and offers the opportunity for in vivo HBV studies [
      • Brown J.J.
      • Parashar B.
      • Moshage H.
      • Tanaka K.E.
      • Engelhardt D.
      • Rabbani E.
      • et al.
      A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice.
      ]. Several hepatic cell lines have also proven useful as in vitro tools for screening and safety testing of drug candidates. For instance, Hc3716-hTERT cells represented the first model for predicting the side-effects of telomere-targeting drugs in normal cells and it was suggested that the Fa2N4 cell line could be used for routine screening during discovery for pregnane X receptor mediated CYP3A4 induction [
      • Waki K.
      • Anno K.
      • Ono T.
      • Ide T.
      • Chayama K.
      • Tahara H.
      Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development.
      ,
      • McGinnity D.F.
      • Zhang G.
      • Kenny J.R.
      • Hamilton G.A.
      • Otmani S.
      • Stams K.R.
      • et al.
      Evaluation of multiple in vitro systems for assessment of CYP3A4 induction in drug discovery: human hepatocytes, pregnane X receptor reporter gene, and Fa2N-4 and HepaRG cells.
      ].

      Conclusions and perspectives

      In vitro expansion of human hepatocytes has gained considerable attention, as it might serve many clinical applications and fundamental research purposes. Prominent examples include the establishment of a bio-artificial human liver device that can be used to bridge the time until liver transplantation is possible and the creation of a liver-based in vitro tool for screening and safety testing of drug candidates. As freshly isolated and cultured mature hepatocytes inherently have very poor growth potential, efforts have focused on strategies to immortalize primary hepatocytes while maintaining their liver-specific functions. The currently available methods include transduction or transfection with prototypical immortalization genes and conditional immortalization by temperature-based regulation, recombinase-based control and transcriptional regulation. Although hepatocyte immortalization has been explored for years, it is still in its infancy since no cell lines with high in vivo-like hepatic functionality are yet available. As already postulated more attention should be paid to culture systems that support differentiation of the immortalized hepatocytes [
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