Isolation and Purification Method of Mouse Fetal Hepatoblasts

  • Luc GailhousteEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 826)


During development, liver precursors constitute a valuable source of pluripotent stem cells that present the ability to differentiate into both a hepatic and biliary lineage. In the present chapter, we report an experimental procedure developed by our group to isolate mouse fetal hepatoblasts (MFHs) with high purity. The method is based on a selective harvesting of the hepatic parenchymal cells from fetuses (E 14.5), followed by the sorting of E-cadherin+ progenitors through the use of magnetic beads and specific antibodies. This protocol allows the isolation of bipotent liver stem cells expressing both hepatic and biliary markers. Primary cultures of purified MFHs can be maintained under proliferation until confluence, leading to promotion of the differentiation process in the presence of hepatotrophic factors. By using a quantitative real-time polymerase chain reaction approach, we show the hepatospecific phenotype and the progressive maturation of MFHs, delineating early (α-fetoprotein), mid (albumin), and late (glucose-6-phosphatase) hepatic markers. Consequently, the model appears to be a valuable cell system for the study of molecular and cellular aspects occurring in hepatic differentiation.

Key words

Mouse fetal hepatoblasts MFHs E-cadherin Cell sorting Bipotent stem cells Hepatic differentiation 



The author thanks Ayako Inoue for her efficient technical assistance, Takahiro Ochiya for his constructive comments on the study, and Takahashi Ryou-u for his advice regarding the cell sorting procedure. This work was supported by a Grant-in-Aid from the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour, Welfare of Japan (H21-001).


  1. 1.
    Ochiya, T., Yamamoto, Y., and Banas, A. (2010) Commitment of stem cells into functional hepatocytes. Differentiation 79, 65–73.PubMedCrossRefGoogle Scholar
  2. 2.
    Fausto, N. (2004) Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39, 1477–1487.PubMedCrossRefGoogle Scholar
  3. 3.
    Roskams, T. (2006) Different types of liver progenitor cells and their niches. J Hepatol 45, 1–4.PubMedCrossRefGoogle Scholar
  4. 4.
    Butz, S., and Larue, L. (1995) Expression of catenins during mouse embryonic development and in adult tissues. Cell Adhes Commun 3, 337–352.PubMedCrossRefGoogle Scholar
  5. 5.
    Dasgupta, A., Hughey, R., Lancin, P., Larue, L., and Moghe, P. V. (2005) E-cadherin synergistically induces hepatospecific phenotype and maturation of embryonic stem cells in conjunction with hepatotrophic factors. Biotechnol Bioeng 92, 257–266.PubMedCrossRefGoogle Scholar
  6. 6.
    Moore, R. N., Dasgupta, A., Rajaei, N., Yarmush, M. L., Toner, M., Larue, L., and Moghe, P. V. (2008) Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes.. Biotechnol Bioeng 101, 1332–1343.PubMedCrossRefGoogle Scholar
  7. 7.
    Wijnhoven, B. P., Dinjens, W. N., and Pignatelli, M. (2000) E-cadherin-catenin cell-cell adhesion complex and human cancer. Br J Surg 87, 992–1005.PubMedCrossRefGoogle Scholar
  8. 8.
    Berx, G., Cleton-Jansen, A. M., Strumane, K., de Leeuw, W. J., Nollet, F., van Roy, F., and Cornelisse, C. (1996) E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 13, 1919–1925.PubMedGoogle Scholar
  9. 9.
    Wei, Y., Van Nhieu, J. T., Prigent, S., Srivatanakul, P., Tiollais, P., and Buendia, M. A. (2002) Altered expression of E-cadherin in hepatocellular carcinoma: correlations with genetic alterations, beta-catenin expression, and clinical features. Hepatology 36, 692–701.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhai, B., Yan, H. X., Liu, S. Q., Chen, L., Wu, M. C., and Wang, H. Y. (2008) Reduced expression of E-cadherin/catenin complex in hepatocellular carcinomas. World J Gastroenterol 14, 5665–5673.PubMedCrossRefGoogle Scholar
  11. 11.
    Nitou, M., Sugiyama, Y., Ishikawa, K., and Shiojiri, N. (2002) Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture. Exp Cell Res 279, 330–343.PubMedCrossRefGoogle Scholar
  12. 12.
    Tanimizu, N., Nishikawa, M., Saito, H., Tsujimura, T., and Miyajima, A. (2003) Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci 116, 1775–1786.PubMedCrossRefGoogle Scholar
  13. 13.
    Dan, Y. Y., Riehle, K. J., Lazaro, C., Teoh, N., Haque, J., Campbell, J. S., and Fausto, N. (2006) Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Proc Natl Acad Sci USA 103, 9912–9917.CrossRefGoogle Scholar
  14. 14.
    Miki, R., Tatsumi, N., Matsumoto, K., and Yokouchi, Y. (2008) New primary culture systems to study the differentiation and proliferation of mouse fetal hepatoblasts. Am J Physiol Gastrointest Liver Physiol 294, 529–539.CrossRefGoogle Scholar
  15. 15.
    Oertel, M., Menthena, A., Chen, Y. Q., Teisner, B., Jensen, C. H., and Shafritz, D. A. (2008) Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver. Gastroenterology 134, 823–832.PubMedCrossRefGoogle Scholar
  16. 16.
    Okabe, M., Tsukahara, Y., Tanaka, M., Suzuki, K., Saito, S., Kamiya, Y., Tsujimura, T., Nakamura, K., and Miyajima, A. (2009) Potential hepatic stem cells reside in EpCAM  +  cells of normal and injured mouse liver. Development 136, 1951–1960.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Division of Molecular and Cellular MedicineNational Cancer Center Research InstituteTokyoJapan

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