Advertisement

Isolation and Characterization of Hepatic Stem Cells, or “Oval Cells,” from Rat Livers

  • Thomas D. Shupe
  • Anna C. Piscaglia
  • Seh-Hoon Oh
  • Antonio Gasbarrini
  • Bryon E. Petersen
Part of the Methods in Molecular Biology book series (MIMB, volume 482)

Abstract

The pace of research on the potential therapeutic uses of liver stem cells or “oval cells” has accelerated significantly in recent years. Concurrent advancements in techniques for the isolation and characterization of these cells have helped fuel this research. Several models now exist for the induction of oval cell proliferation in rodents. Protocols for the isolation and culture of these cells have evolved to the point that they may be set up in any laboratory equipped for cell culture. The advent of magnetic cell sorting has eliminated reliance on expensive flow cytometric sorting equipment to generate highly enriched populations of oval cells. Our laboratory has had much success in using the oval cell surface marker Thy-1 in combination with magnetic sorting to produce material suitable for testing the influence of a myriad of chemical signaling molecules on the oval cell phenotype. This chapter will describe our basic strategy for oval cell induction and isolation. Additionally, two in vitro procedures are described which the reader may find useful in the early stages of developing an oval cell research project.

Key words

Liver stem cells oval cells 2-acetylaminofluorene (2AAF) partial hepatectomy (PH) collagenase perfusion simple gravity enrichment immunomagnetic sorting (IMS) flow cytometric sorting (FACS) proliferation assay migration assay 

References

  1. 1.
    Newsome, P.N., Hussain, M.A., Theise, N.D. (2004) Hepatic oval cells: helping redefine a paradigm in stem cell biology. Curr Top Dev Biol 61, 1–28.CrossRefPubMedGoogle Scholar
  2. 2.
    Oh, S.H., Hatch, H.M., Petersen, B.E. (2002) Hepatic oval ‘stem' cell in liver regeneration. Semin Cell Dev Biol 13, 405–409.CrossRefPubMedGoogle Scholar
  3. 3.
    Petersen, B.E. (2001) Hepatic “stem” cells: coming full circle. Blood Cells Mol Dis 27, 590–600.CrossRefPubMedGoogle Scholar
  4. 4.
    Piscaglia, A.C., Di Campli, C., Gasbarrini, G., Gasbarrini, A. (2003) Stem cells: new tools in gastroenterology and hepatology. Dig Liver Dis 35, 507–514.CrossRefPubMedGoogle Scholar
  5. 5.
    Lowes, K.N., Croager, E.J., Olynyk, J.K., Abraham, L.J., Yeoh, G.C. (2003) Oval cell-mediated liver regeneration: Role of cytokines and growth factors. J Gastroenterol Hepatol 18, 4–12.CrossRefPubMedGoogle Scholar
  6. 6.
    Libbrecht, L., Desmet, V., Van Damme, B., Roskams, T. (2000) Deep intralobular extension of human hepatic ‘progenitor cells ‘correlates with parenchymal inflammation in chronic viral hepatitis: can 'progenitor cells' migrate? J Pathol 192, 373–378.CrossRefPubMedGoogle Scholar
  7. 7.
    Mohle, R., Bautz, F., Denzlinger, C., Kanz, L. (2001) Transendothelial migration of hematopoietic progenitor cells. Role of chemotactic factors. Ann N Y Acad Sci. 938, 26–34; discussion 34–35.CrossRefPubMedGoogle Scholar
  8. 8.
    Petersen, B.E., Zajac, V.F., Michalopoulos, G.K. (1997) Bile ductular damage induced by methylene dianiline inhibits oval cell activation. Am J Pathol 151, 905–909.PubMedGoogle Scholar
  9. 9.
    Petersen, B.E., Zajac, V.F., Michalopoulos, G.K. (1998) Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats. Hepatology 27, 1030–1038.CrossRefPubMedGoogle Scholar
  10. 10.
    Alison, M.R., Golding, M., Sarraf, C.E., Edwards, R.J., Lalani, E.N. (1996) Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells. Gastroenterology 110, 1182–1190.CrossRefPubMedGoogle Scholar
  11. 11.
    Evarts, R.P., Hu, Z., Omori, N., Omori, M., Marsden, E.R., Thorgeirsson, S.S. (1996) Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers. Carcinogenesis 17, 2143–2151.CrossRefPubMedGoogle Scholar
  12. 12.
    Higgins, G. M., and Anderson, R. M. (1931) Experimental pathology of the liver. Arch Pathol 12, 186–201.Google Scholar
  13. 13.
    Petersen, B.E., and Hatch, H.M. (2002) Stem Cell Culture: Liver Stem Cells. Methods of Tissue Engineering: 429–437. Academic Press. 2002Google Scholar
  14. 14.
    Schmitz, B., et al. (1994) Magnetic activated cell sorting (MACS)--a new immunomagnetic method for megakaryocytic cell isolation: comparison of different separation techniques. Eur J Haematol 52, 267–275.CrossRefPubMedGoogle Scholar
  15. 15.
    Von Schönfeldt, V., Krishnamurthy, H., Foppiani, L., Schlatt, S. (1999) Magnetic Cell Sorting Is a Fast and Effective Method of Enriching Viable Spermatogonia from Djungarian Hamster, Mouse, and Marmoset Monkey Testes. Biology Reprod 61, 582–589.CrossRefGoogle Scholar
  16. 16.
    Campbell, D.G., Gagnon, J., Reid, K.B.M., Williams, A.F. (1981) Rat brain Thy-1 glycoprotein. The amino acid sequence, disulphide bonds and an unusual hydrophobic region. Biochem J 195, 15–30.PubMedGoogle Scholar
  17. 17.
    Hermans, M.H.A., and Opstelten, D. (1991) In situ visualization of hemotopoietic cell subsets and stromal elements in rat and mouse bone marrow by immunostaining of frozen sections. J Histochem Cytochem 39, 1627–1634.PubMedGoogle Scholar
  18. 18.
    Garnett, D., Barclay, A.N., Carmo, A.M., Beyers, A.D. (1993) The association of the protein tyrosine kinases p56lck and p60fyn with the glycosyl phosphatidylinositol anchored proteins Thy-1 and CD48 in rat thymocytes is dependent on the state of cellular activation. Eur J Immunol 23, 2540–2544.CrossRefPubMedGoogle Scholar
  19. 19.
    Crook, K., and Hunt, S.V. (1996) Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82. Dev Immunol 4, 235–246.PubMedGoogle Scholar
  20. 20.
    Ishizu, A., et al. (1995) Thy-1 induced on rat endothelium regulates vascular permeability at sites of inflammation. Int Immunol 7, 1939–1947.CrossRefPubMedGoogle Scholar
  21. 21.
    Petersen, B.E., Goff, J.P., Greenberger, J.S., Michalopoulos, G.K. (1998) Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27, 433–445.CrossRefPubMedGoogle Scholar
  22. 22.
    Mason, D.W., and Williams, A.M. (1980) The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem J 187, 1–20.PubMedGoogle Scholar
  23. 23.
    Hatch, H.M., Zheng, D., Jorgensen, M.L., Petersen, B.E. (2002) SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 4, 339–51.CrossRefPubMedGoogle Scholar
  24. 24.
    Jung, Y., Oh, S.H., Zheng, D., Shupe, T.D., Witek, R.P., Petersen, B.E. (2006) A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells. Lab Invest 86, 477–489.CrossRefPubMedGoogle Scholar
  25. 25.
    Piscaglia, A.C., Shupe, T.D., Oh, S., Gasbasrini, A., Petersen, B.E. (2007) Granulocyte-Colony Stimulating Factor Promotes Liver Repair and Induces Oval Cell Migration and Proliferation in Rats. Gastroenterology 133, 619–631.Google Scholar
  26. 26.
    Stolz, D.B., and Michalopoulos, G.K. (1997) Synergistic enhancement of EGF, but not HGF, stimulated hepatocyte motility by TGF-beta 1 in vitro. J Cell Physiol 170, 57–68.CrossRefPubMedGoogle Scholar
  27. 27.
    Weissman, I.L. (2002) Stem cells - scientific, medical, and political issues. New Eng J Med 346, 1576–1579.CrossRefPubMedGoogle Scholar
  28. 28.
    Marshall, E. (2000) The business of stem cells. Science 287, 1419.CrossRefPubMedGoogle Scholar
  29. 29.
    Winstor, R. (2001) Embryonic stem cell research. The case for… Nat Med 7, 396–397.CrossRefGoogle Scholar
  30. 30.
    Ferber, S. (2000) Can we create new organs from our own tissues? Isr Med Assoc J 2(suppl 2), 32–36.PubMedGoogle Scholar
  31. 31.
    Grompe, M. (2001) Liver repopulation for the treatment of metabolic diseases. J Inherit Metab Dis 24, 231–244.CrossRefPubMedGoogle Scholar
  32. 32.
    Paul, S., and Regulier, E. (2000) Molecular basis of oncogenesis. Ann Biol Clin 59, 393–402.Google Scholar
  33. 33.
    Bach, S.P., Renehan, A.G., Potten, C.S. (2000) Stem cells: the intestinal cell as a paradigm. Carcinogenesis 21, 469–476.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Thomas D. Shupe
    • 1
  • Anna C. Piscaglia
    • 2
    • 3
  • Seh-Hoon Oh
    • 1
  • Antonio Gasbarrini
    • 3
  • Bryon E. Petersen
    • 2
    • 4
  1. 1.Department of Pathology, Immunology and Laboratory Medicine, College of MedicineUniversity of FloridaGainesvilleUSA
  2. 2.Department of Pathology, Immunology and Laboratory Medicine, College of MedicineUniversity of FloridaGainesville
  3. 3.Department of Internal Medicine and GastroenterologyCatholic University of RomeItaly
  4. 4.Program for Stem Cell Biology, University of Florida - Shands Cancer CenterGainesvilleUSA

Personalised recommendations