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. Author manuscript; available in PMC: 2015 Sep 23.
Published in final edited form as: Methods Mol Biol. 2015;1250:53–76. doi: 10.1007/978-1-4939-2074-7_4

Table 2. Overview of conditional immortalization strategies, hepatic functionality and in vitro applications of growth-controlled human adult and fetal hepatic cell lines.

(A1AT, α1-antitrypsin; AFP, α-fetoprotein; ALB, albumin; ASGP(R), asialoglycoprotein (receptor); Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog; C/EBP, Ccaat-enhancer-binding protein; CD, cluster of differentiation; CK, cytokeratin; CYP, cytochrome P450; GS, glutamine synthetase; GST, gluthatione S-transferase; HBCF, human blood coagulation factor; HNF, hepatocyte nuclear factor; hTERT, human telomerase reverse transcriptase; mRNA, messenger ribonucleic acid; PT, prothrombin; SV40 Tag, simian virus 40 large T antigen; TF, transferrin; (bil-)UGT, (bilirubin-)uridinediphosphate-glucuronosyltransferase).

Adult hepatic cell line
Cell line Immortalization strategy Hepatic functionality of conditional immortalized cells In vitro applications Reference
16T-3 Retroviral vector Reverted 16T-3 cells:
  • -

    Show enhanced mRNA levels of transcriptional factors, C/EBPα and HNF4α as well as increased mRNA expression of hepatocyte-specific genes, including ALB, GST-Π, HBCF-X, bil-UGT, CYP3A4, GS and ASGPR.

  • -

    Possess increased ALB production and lidocaine metabolism, though at lower levels than normal human hepatocytes.

 (43)
hTERT
Tamoxifen-mediated self-excision (Cre-LoxP)
HepLi-4 Retroviral vector Reverted HepLi-4 cells:
  • -

    Express similar GS and somewhat lower UGT1A1 mRNA levels than adult human liver. ALB and GST-Π mRNA levels are extremely lower or higher, respectively, compared to the human liver. This indicates that HepLi-4 cells are not fully differentiated after reversion.

 (26)
SV40 Tag
Tamoxifen-mediated self-excision (Cre-LoxP)
HLTC-7/ -11/ -15/ -17/ -19 Retroviral vector
  • -

    Grow as islands or sheets of cuboidal cells (HLTC-17) or display a more dispersed cuboidal-elongated morphology (HLTC-7/-11/-15/-19).

  • -

    Secrete fibrinogen at fairly constant rate in all tested cell lines at permissive (33,5°C) and non-permissive (39,5°C) temperature.

  • -

    Exhibit no ALB, AFP, A1AGP or secretion in any cell line at both temperatures.

  • -

    Cell lines HLTC-7,-15 and -19, produce A1AT at permissive temperature. However, at non-permissive temperature the secretion of A1AT is upregulated or become detectable in all the cell lines.

  • -

    Cell lines HLTC-17 and -11 possess no CYP activity at any temperature even after induction and stain positive for ALB, CK18, CK7, CK19 and vimentin, but negative for CK8, with almost identical patterns at both temperatures.



  • ➢

    The results indicate progressive phenotypic instability and loss of differentiated functions. Conversion to the non-permissive temperature does only allow significant expression of a limited repertoire of differentiated functions by the immortalized human hepatocytes.

 (28)
SV40 Tag
Temperature-based regulation
IHH10(.3)/12 Lentiviral vector
  • -

    Display morphology reminiscent of differentiated hepatocytes.

  • -

    Express ALB, A1AT, ASGPR and CYP450 mRNA levels.

  • -

    Secrete liver-specific proteins, ALB and fibrinogen, at levels similar to Huh-7 cells but lower than primary hepatocytes. The IHH12 cell line do only produce fibrinogen after de-immortalization, suggesting the acquirement of a higher differentiation status in this setting. However, Cre-recombinase treatment of IHH12 cells does not significantly improve the production of ALB.

  • -

    Possess inducible CYP1A1/2 activity.

 A novel in vitro model to investigate the mechanisms and consequences of lipid accumulation in hepatocytes, independently of insulin resistance. (19, 87)
SV40 Tag + hTERT (IHH10)
or
SV40 Tag + hTERT + Bmi-1 (IHH12)
Recombinase- based control (Cre-LoxP)
NKNT-3 Retroviral vector
  • -

    Display morphological characteristics of liver parenchymal cells and look more differentiated after reversion.

  • -

    Express bil-UGT, GS and GST-Π mRNA levels, which increased substantially after reversion. Contradicting results are published regarding expression of ALB and HBCF-X mRNA levels. One paper demonstrates that ALB and HBCF-X mRNA are newly introduced in the reverted cells whereas several other papers already report expression of these genes and ASGPR mRNA in non-reverted cells. Nevertheless, although reversion does stimulate differentiation, mRNA levels of ALB, A1AT and TF were maximally 0.1% of primary human hepatocytes.



  • ➢

    Additional experiments reveal that introduction of p21 into human immortalized hepatocytes can increase ALB expression and induce a differentiated morphology.

  • ➢

    Co-cultivation with immortalized hepatic stellate cells increases urea synthesis and protein expression of CYP3A4/2C9.

 Used in combination with HCV like particles as a model system for studying viral binding and entry. (6, 29,52, 71, 79)
SV40 Tag
Recombinase- based control (Cre-loxP)
YOCK-13 Retroviral vector
  • -

    Display morphological characteristics of normal human hepatocytes.

  • -

    Express markers of hepatocytic differentiation including ALB, ASGPR, bil-UGT, CYP3A4, GS, GST-Π, and HBCF-X.



  • ➢

    The YOCK-13 hepaticcell line is derived from the reversible immortalized human hepatic cell line, TTNT-16-3, by co-expression of modified insulin.

 (42)
hTERT
Tamoxifen-mediated self-excision (Cre-LoxP)
Fetal hepatic cell lines
Cell line Immortalization strategy Hepatic functionality of conditional immortalized cells In vitro applications Reference
cBAL111 Lentiviral vector
  • -

    Express relatively high mRNA levels of immature markers, GST-Π and AFP, and very low mRNA levels of mature markers, ALB, A1AT and TF. Transcript levels of HNF4α increase after prolonged culturing.

  • -

    Stain positive for GS, ALB, CK18, CK19, vimentin and the progenitor cell marker CD146 but display CK18 in a pattern characteristic of dedifferentiated human hepatocytes.

  • -

    Produce urea and ALB, though at lower levels than mature human hepatocytes.

  • -

    Retain no CYP1A2 and 3A4 activity (no elimination of lidocaine) but are able to eliminate galactose.



  • ➢

    cBAL111cells resemble cells with progenitor characteristics rather than fully differentiated hepatocytes. However, there is a trend of increased and decreased expression of mature and immature markers, respectively, with culture time.

 (13)
hTERT
Transcriptional regulation (Tet-on approach)
HepCL Retroviral vector
  • -

    Display morphological characteristics of liver parenchymal cells.

  • -

    Stain positive for ALB, CK18 and CK19.

  • -

    Produce amounts of ALB and urea comparable to those of unmodified primary human fetal hepatocytes.

 (27)
SV40 Tag
Temperature-based regulation