Advertisement

Development of Immortalized Hepatocyte-Like Cells from hMSCs

  • Adisak Wongkajornsilp
  • Khanit Sa-ngiamsuntorn
  • Suradej HongengEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 826)

Abstract

Clones of hepatocyte-like cells were reproducibly generated from human mesenchymal stem cells immortalized with a combined transduction of both Bmi-1 and TERT genes. These hepatocyte-like cells contained selective markers and several functional properties of hepatocytes, yet still carried proliferative potential. These cells had cuboidal morphology and arranged themselves as cord-like structure in culture. The cloned cells deposited glycogen and actively synthesized albumin. The basal expressions of CYP450 isozymes was observed, albeit only 10–20% that of primary hepatocytes. These expressions were promptly increased upon the addition of rifampicin, a known enzyme inducer. These hepatocyte-like cells may serve as a close alternative to the use of primary hepatocytes for in vitro studies.

Key words

Hepatocyte-like cell hMSC Cell immortalization Hepatocyte differentiation CYP450 Drug metabolism Toxicology 

References

  1. 1.
    Gomez-Lechon MJ, Donato MT, Castell JV, Jover R. Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Curr Drug Metab. 2004 Oct;5(5):443–62.PubMedCrossRefGoogle Scholar
  2. 2.
    Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. 2002 Jun;32(6):505–20.PubMedCrossRefGoogle Scholar
  3. 3.
    Gomez-Lechon MJ, Castell JV, Donato MT. Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo. Chem Biol Interact. 2007 May 20;168(1):30–50.PubMedCrossRefGoogle Scholar
  4. 4.
    Guguen-Guillouzo C, Corlu A, Guillouzo A. Stem cell-derived hepatocytes and their use in toxicology. Toxicology. 2010 Mar 30;270(1):3–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Khetani SR, Bhatia SN. Microscale culture of human liver cells for drug development. Nat Biotechnol. 2008 Jan;26(1):120–6.PubMedCrossRefGoogle Scholar
  6. 6.
    Youdim KA, Tyman CA, Jones BC, Hyland R. Induction of cytochrome P450: assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay. Drug Metab Dispos. 2007 Feb;35(2):275–82.PubMedCrossRefGoogle Scholar
  7. 7.
    Sinz M, Wallace G, Sahi J. Current industrial practices in assessing CYP450 enzyme induction: preclinical and clinical. AAPS J. 2008 Jun;10(2):391–400.PubMedCrossRefGoogle Scholar
  8. 8.
    Banas A, Yamamoto Y, Teratani T, Ochiya T. Stem cell plasticity: learning from hepatogenic differentiation strategies. Dev Dyn. 2007 Dec;236(12):3228–41.PubMedCrossRefGoogle Scholar
  9. 9.
    Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999 Apr 2;284(5411):143–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem. 2000 Mar 31;275(13):9645–52.PubMedCrossRefGoogle Scholar
  11. 11.
    Nagai A, Kim WK, Lee HJ, Jeong HS, Kim KS, Hong SH, et al. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow. PLoS One. 2007;2(12):e1272.PubMedCrossRefGoogle Scholar
  12. 12.
    Ong SY, Dai H, Leong KW. Hepatic differentiation potential of commercially available human mesenchymal stem cells. Tissue Eng. 2006 Dec;12(12):3477–85.PubMedCrossRefGoogle Scholar
  13. 13.
    Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology. 2007 Jul;46(1):219–28.PubMedCrossRefGoogle Scholar
  14. 14.
    Zemel R, Bachmetov L, Ad-El D, Abraham A, Tur-Kaspa R. Expression of liver-specific markers in naive adipose-derived mesenchymal stem cells. Liver Int. 2009 Oct;29(9):1326–37.PubMedCrossRefGoogle Scholar
  15. 15.
    Yamamoto Y, Banas A, Murata S, Ishikawa M, Lim CR, Teratani T, et al. A comparative analysis of the transcriptome and signal pathways in hepatic differentiation of human adipose mesenchymal stem cells. FEBS J. 2008 Mar;275(6):1260–73.PubMedCrossRefGoogle Scholar
  16. 16.
    Kulkarni JS, Khanna A. Functional hepatocyte-like cells derived from mouse embryonic stem cells: a novel in vitro hepatotoxicity model for drug screening. Toxicol In Vitro. 2006 Sep;20(6):1014–22.PubMedCrossRefGoogle Scholar
  17. 17.
    Ek M, Soderdahl T, Kuppers-Munther B, Edsbagge J, Andersson TB, Bjorquist P, et al. Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem Pharmacol. 2007 Aug 1;74(3):496–503.PubMedCrossRefGoogle Scholar
  18. 18.
    Unger C, Gao S, Cohen M, Jaconi M, Bergstrom R, Holm F, et al. Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells. Hum Reprod. 2009 Oct;24(10):2567–81.PubMedCrossRefGoogle Scholar
  19. 19.
    Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc. 2009;4(4):495–505.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Adisak Wongkajornsilp
    • 1
  • Khanit Sa-ngiamsuntorn
    • 1
  • Suradej Hongeng
    • 2
    Email author
  1. 1.Department of Pharmacology, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  2. 2.Department of Pediatrics, Faculty of Medicine, Ramathibodi HospitalMahidol UniversityBangkokThailand

Personalised recommendations