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Putative liver progenitor cells: conditions for long-term survival in culture

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Abstract

Oval cells proliferate extensively in the livers of animals exposed to oncogenic insults, are bipotent and are believed to be related to the so far unidentified liver stem cell. In normal liver, cells antigenica lly related to oval cells and expressing liver and epithelial markers are considered to be liver progenitor cells. We isolated, by fluorescence-activated cell sorting or magnetic bead sorting, cells expressing the oval cell antigens OC.2 or OC.3 from the liver of normal newborn or day 12 embryonal age rats. Magnetic bead sorting of positive cells was as efficient as fluorescence-activated cell sorting. A two-chamber culture system was devised in which cells were plated onto transwell filters coated with type IV collagen and cultured in a serum-free Ham's F12 medium supplemented with free fatty acids and bovine serum albumin. Under these conditions, cells remained viable for up to 6 weeks and their antigenic phenotype was unchanged throughout. Approximately 30% of sorted cells expressed epithelial and/or liver-specific markers. Growth factors mitogenic for epithelial cells and hepatocytes did not elicit cell proliferation. These results provide an important background for further studies designed to determine the biological significance of OC.2+ and OC.3+ cells in normal liver, to test the liver stem cell hypothesis and to develop protocols for the expansion in vitro of normal liver progenitors.

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References

  • Agelli, M. & Halay, E.D. (1995) Flow cytometry and monoclonal antibodies identify normal liver cell populations antigenically related to oval cells. Eur. J. Histochem. 39, 1–8.

    Google Scholar 

  • Ailenberg, M., Tung, P.S., Pelletier, M. & Fritz, I.B. (1988) Modulation of Sertoli cell functions in two-chamber assembly by peritubular cells and extracellular matrix. Endocrinology 122, 2604–12.

    Article  PubMed  CAS  Google Scholar 

  • Alvarez, E. & Blazquez, E. (1987) Lack of insulin effect on its own receptors in fetal rat hepatocytes. Horm. Metab. Res. 19, 458–63.

    Article  PubMed  CAS  Google Scholar 

  • Barclay, A.N., Jackson, D.I., Willis, A.C. & Williams, A.F. (1987) Lymphocyte specific heterogeneity in the rat leucocyte common antigen (T200) is due to differences in polypeptide sequences near the NH2-terminus. EMBO J. 6, 1259–64.

    PubMed  CAS  Google Scholar 

  • Baribault, H., Leroux-Nicollet, I. & Marceau, N. (1985) Differential responsiveness of cultured suckling and adult hepatocytes to growth-promoting factors: entry into S phase and mitosis. J. Cell Physiol. 122, 105–12.

    Article  PubMed  CAS  Google Scholar 

  • Berry, M.N. & Friend, D.S. (1969) High-yield preparation of isolated rat liver parenchymal cells. A biochemical and fine structural study. J. Cell Biol. 43, 506–20.

    Article  PubMed  CAS  Google Scholar 

  • Billet, E.E., Gunn, B. & Mayer, R.J. (1984) Characterization of two monoclonal antibodies obtained after immunization with human liver mitochondrial membrane preparations. Biochem. J. 221, 765–776.

    Google Scholar 

  • Bralet, M.-P., Branchereau, S., Brechot, C. & Ferry, N. (1994) Cell lineage study in the liver using retroviral mediated gene transfer. Evidence against the streaming of hepatocytes in normal liver. Am. J. Pathol. 144, 896–905.

    PubMed  CAS  Google Scholar 

  • Bucher, N.L.R. & McGowan, J.A. (1979) Regulatory mechanisms in liver regeneration. In Liver and Biliary Disease: A Pathobiological Approach. (edited by Wright, R., Alberti, G.G.M.M., Karsan, S. and Millward-Sadler, H.) pp. 210–227. Philadelphia, PA, USA: W. B. Saunders & Co.

    Google Scholar 

  • Carson, D.D., Tang, J.P., Julian, J. & Glasser, S.R. (1988) Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells. J. Cell Biol. 107, 2425–35.

    Article  PubMed  CAS  Google Scholar 

  • Cascio, S. & Zaret, K.S. (1991) Hepatocyte differentiation initiates during endodermal-mesenchymal interations prior to liver formation. Development 113, 217–25.

    PubMed  CAS  Google Scholar 

  • Chessebeuf, M. & Padieu, P. (1984) Rat liver epithelial cell cultures in a serum-free medium: primary cultures and derived cell lines expressing differentiated functions. In Vitro 20, 780–95.

    Article  PubMed  CAS  Google Scholar 

  • Cruise, J.L., Houck, K.A. & Michalopoulos, G.K. (1985) Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha1-adrenoreceptor by norepinephrine. Science 227, 749–51.

    Article  PubMed  CAS  Google Scholar 

  • Dallman, M.J., Mason, D.W. & Webb, M. (1982) The role of host and donor cells in the rejection of skin allografts by T cell-deprived rats injected with syngenic T cells. Eur. J. Immunol. 12, 511–18.

    Article  PubMed  CAS  Google Scholar 

  • De Juan, C., Benito, M., Alvarez, A. & Fabrega, I. (1992) Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones. Exp. Cell Res. 202, 495–500.

    Article  PubMed  CAS  Google Scholar 

  • Dello Sbarba, P., Cipolleschi, M.G. & Olivotto, M. (1987) Hemopoietic progenitor cells are sensitive to the cytostatic effect of piruvate. Exp. Hematol. 15, 137–42.

    PubMed  CAS  Google Scholar 

  • Dexter, T.M., Allen, T.D. & Lajtha, L.G. (1976) Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell Physiol. 91, 335–44.

    Article  Google Scholar 

  • Dijkstra, C.D., Dopp, E.A., Joling, P. & Kraal, G. (1985) The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology 54, 589–99.

    PubMed  CAS  Google Scholar 

  • Dunsford, H.A., Maset, R., Solman, J. & Sell, S. (1985) Connection of duct-like structures induced by a chemical hepatocarcinogen to portal bile ducts in the rat liver detected by injection of bile ducts with a pigmented barium gelatin medium. Am. J. Pathol. 118, 218–24.

    PubMed  CAS  Google Scholar 

  • Enat, R., Jefferson, D.M., Ruiz-Opazo, N., Gatmaitan, Z., Leinwand, L.A. & Reid, L.M. (1984) Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc. Natl Acad. Sci. USA 81, 1411–15.

    Article  PubMed  CAS  Google Scholar 

  • Evarts, R.P., Nagy, P., Marsden, E.R. & Thorgeirsson, S.S. (1987a) A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8, 1737–40.

    Article  PubMed  CAS  Google Scholar 

  • Evarts, R.P., Nagy, P., Marsden, E.R. & Torgeirsson, S.S. (1987b) In situ hybridization studies on expression of albumin and ·-fetoprotein during the early stage of neoplastic transformation in rat liver. Cancer Res. 47, 5469–75.

    PubMed  CAS  Google Scholar 

  • Evarts, R.P., Nagy, P., Nakatsukasa, H., Marsden, E. & Thorgeirsson, S.S. (1989) In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res. 49, 1541–47.

    PubMed  CAS  Google Scholar 

  • Evarts, R.P., Hu, Z., Fujio, K., Marsden, E.R. & Thorgeirsson, S.S. (1993) Activation of hepatic stem cell compartment in the rat: role of transforming growth factor alpha, hepatocyte growth factor, and acidic fibroblast growth factor in early proliferation. Cell Growth Differ. 4, 555–61.

    PubMed  CAS  Google Scholar 

  • Farber, E. (1965) Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3-methyl-4-dimethylamino azobenzene. Cancer Res. 16, 142–49.

    Google Scholar 

  • Farber, E. (1982) Chemical carcinogenesis: A biological perspective. Am. J. Pathol. 106, 292–96.

    Google Scholar 

  • Fausto, N. (1990) Hepatocyte differentiation and liver progenitor cells. Curr. Opin. Cell Biol. 2, 1036–42.

    Article  PubMed  CAS  Google Scholar 

  • Fausto, N., Thompson, N.L. & Braun, L. (1987) Purification and culture of oval cells from rat liver. In Cell Separation, Methods and Selected Applications (edited by Pretlow, II, T.G. and Pretlow, T.P.). pp. 45–77. New York, NY, USA: Academic Press Inc.

    Google Scholar 

  • Fausto, N., Lemire, J.M. & Shiojiri, N. (1993) Cell lineages in hepatic development and the identification of progenitor cells in normal and injured liver. Proc. Soc. Exp. Biol. Med. 204, 237–41.

    PubMed  CAS  Google Scholar 

  • Gatmaitan, Z., Jefferson, D.N., Ruiz-Opazo, N., Biempica, L., Arias, I.M., Dwadas, G., Leinwand, L.A. & Reid, L.M. (1983) Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrate. J. Cell Biol. 97, 1179–90.

    Article  PubMed  CAS  Google Scholar 

  • Germain, L., Blouin, M.J. & Marceau, N. (1988) Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, ·-fetoprotein, albumin, and cell-surface-exposed components. Cancer Res. 48, 4909–18.

    PubMed  CAS  Google Scholar 

  • Grisham, J.W. (1980) Cell types in long-term propagable cultures of rat liver. Ann. NY Acad. Sci. 349, 128–37.

    Article  PubMed  CAS  Google Scholar 

  • Haeuptle, M.T., Suard, Y.L.M., Bogenmann, E., Reggio, H., Racine, L. & Kraehrnbuhl, J.P. (1983) Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture. J. Cell Biol. 96, 1425–34.

    Article  PubMed  CAS  Google Scholar 

  • Hixson, D.C. & Alison, J.P. (1985) Monoclonal antibodies recognizing oval cells induced in the liver of rats by N-2-fluorenyl acetamide or ethionine in a choline-deficient diet. Cancer Res. 45, 3750–60.

    PubMed  CAS  Google Scholar 

  • Hixson, D.C., Faris, R.A. & Thompson, N.L. (1990) An antigenic portrait of the liver during carcinogenesis. Pathobiology 58, 65–77.

    Article  PubMed  CAS  Google Scholar 

  • Hsia, C.C., Evarts, R.P., Natatsukasa, H., Marsden, E.R. & Thorgeirsson, S.S. (1992) Occurrance of oval-type cells in hepatitis B virus-associated human hepatocarcinogenesis. Hepatology 16, 1327–33.

    Article  PubMed  CAS  Google Scholar 

  • Ishii, M., Vroman, B. & La Russo, N.F. (1989) Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97, 1236–47.

    PubMed  CAS  Google Scholar 

  • Lombardi, B. (1982) On the nature, properties and significance of oval cells. In Recent Trends in Chemical Carcinogenesis, Vol. 1. (edited by Pani, F., Feo, F., and Columbano, A.), pp. 36–56. Cagliari, Italy: ESA.

    Google Scholar 

  • Martinez-Hernandez, A. & Amenta, P.S. (1993) Morphology, localization and cellular origin of hepatic extracellular matrix. In Extracellular Matrix: Chemistry, Biology and Pathobiology (edited by Zern, M. and Reid, L.M.), pp. 255–327. New York: Marcer Dekker.

    Google Scholar 

  • Michalopoulos, G. & Pitot, H.C. (1975) Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94, 70–8.

    Article  PubMed  CAS  Google Scholar 

  • Nagy, P., Bisgaard, H.C. & Thorgeirsson, S.S. (1994) Expression of hepatic transcription factors during liver development and oval cell differentiation. J. Cell Biol. 126, 223–33.

    Article  PubMed  CAS  Google Scholar 

  • Onda, H. (1976) Immunohistochemical studies on alpha-1-fetoprotein and alpha-1-acid glycoprotein during azo dye hepatocarcinogenesis in rats. GANN 67, 253–62.

    PubMed  CAS  Google Scholar 

  • Parry, G., Cullen, B., Kaetzel, C.S., Kramer, R. & Moss, L. (1987) Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences. J. Cell Biol. 105, 2043–51.

    Article  PubMed  CAS  Google Scholar 

  • Paterson, D.J. (1987) The MRC OX-44 antigen marks a functionally relevant subset among rat thymocytes. J. Exp. Med. 165, 1–13.

    Article  PubMed  CAS  Google Scholar 

  • Plenat, F., Braun, L. & Fausto, N. (1988) Demonstration of glucose-6-phosphatase and peroxisomal catalase activity by ultrastructural cytochemistry in oval cells from livers of carcinogen-treated rats. Am. J. Pathol. 130, 91–102.

    PubMed  CAS  Google Scholar 

  • Potter, V.R. (1981) The present status of the blocked ontogeny hypothesis of neoplasia: The thalassemia connection. Oncodevelopemental Biol. Med. 2, 243–266.

    CAS  Google Scholar 

  • Reid, M.L. (1990) Stem cell biology, hormone/matrix synergies and liver differentiation. Curr. Opin. Cell Biol. 2, 121–30.

    Article  PubMed  CAS  Google Scholar 

  • Robinson, A.P., White, T.M. & Mason, D.W. (1986) Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3. Immunology 57, 239–47.

    PubMed  CAS  Google Scholar 

  • Rutenberg, A.M., Kim, H. & Fishbein, J.W. (1969) Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J. Histochem. Cytochem. 17, 517–26.

    Google Scholar 

  • Sato, J.D., Kawamoto, T., Le, A.D., Mendelson, J., Polikoff, J. & Sato, G.H. (1983) Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol. Biol. Med. 1, 511–29.

    PubMed  CAS  Google Scholar 

  • Sell, S. (1990) Is there a liver stem cell? Cancer Res. 50, 3811–15.

    PubMed  CAS  Google Scholar 

  • Sell, S. & Dunsford, H.A. (1989) Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma. Am. J. Pathol. 134, 1347–63.

    PubMed  CAS  Google Scholar 

  • Sell, S. & Pierce, G.B. (1994) Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab. Invest. 70, 6–22.

    PubMed  CAS  Google Scholar 

  • Sell, S., Osborn, K. & Leffert, H.L. (1981) Autoradiography of ‘oval’ cells appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet. Carcinogenesis 2, 71–4.

    Article  Google Scholar 

  • Shiojiri, N. (1981) Enzymo-and immuno-cytochemical analyses of the differentiation of liver cells in the prenatal mouse. J. Embryol. Exp. Morphol. 62, 139–52.

    PubMed  CAS  Google Scholar 

  • Sigal, S.H., Brill, S., Reid, L.M., Zvibel, I., Gupta, S., Hixson, D., Faris, R. & Holst, P.A. (1994) Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption (‘panning’) and fluorescent-activated cell sorting. Hepatology 19, 999–1006.

    PubMed  CAS  Google Scholar 

  • Sigal, S.H., Gupta, S., Gebhard, D.F., Jr., Holst, P.A., Neufeld, D. & Reid, L.M. (1995) Evidence for a terminal differentiation process in the rat liver. Differentiation 59, 35–42.

    Article  PubMed  CAS  Google Scholar 

  • Travis, J. (1993) The search for liver stem cells picks up. Science 259, 1829.

    Article  PubMed  CAS  Google Scholar 

  • Vassy, J., Kraemer, M., Chalumeau, M.T. & Foucrier, J. (1988) Development of the fetal liver: ultrastructural and serological study of hepatocytes. Cell. Diff. 24, 9–24.

    Article  CAS  Google Scholar 

  • Webber, E.M., Godowski, P.J. & Fausto, N. (1994) In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology 19, 489–97.

    Article  PubMed  CAS  Google Scholar 

  • Wilson, J.W. & Leduc, E.H. (1958) Role of cholangioles in restoration of the liver of the mouse after dietary injury. J. Pathol. Bacteriol. 76, 441–49.

    Article  PubMed  CAS  Google Scholar 

  • Wu, J.C., Merlino, G., Cveklova, K., Mosinger, B., Jr., & Nelson, F. (1994) Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that over-express transforming growth factor ·. Cancer Res. 54, 5964–73.

    PubMed  CAS  Google Scholar 

  • Yang, L., Faris, R.A., Hixson, D.C. (1993) Characterization of a mature bile duct antigen expressed on a subpopulation of biliary ductular cells but absent from oval cells. Hepatology 18, 357–66.

    PubMed  CAS  Google Scholar 

  • Yokoyama, S., Satoh, M. & Lombardi, B. (1986) Bile ductular cells and the phenotypic heterogeneity of the population of hepatic non-parenchymal epithelial cells induced in rats by chemical carcinogens. Carcinogenesis 7, 1215–19.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. The Liver Center, The Albert Einstein College of Medicine, Bronx, New York, USA

    Maria Agelli, Elaine D. Halay & Lola M. Reid

  2. Department of Microbiology and Immunology, The Albert Einstein College of Medicine, Bronx, New York, USA

    Maria Agelli, Elaine D. Halay & Lola M. Reid

  3. Developmental Biology and Cancer, The Albert Einstein College of Medicine, Bronx, New York, USA

    Persio Dello Sbarba

  4. Department of Pediatric Research, Rhode Island Hospital, Providence, Rhode Island, USA

    Ronald A. Faris

  5. Medical Oncology, Rhode Island Hospital, Providence, Rhode Island, USA

    Douglas E. Hixson

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  1. Maria Agelli
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  2. Elaine D. Halay
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  3. Lola M. Reid
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  4. Persio Dello Sbarba
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Agelli, M., Halay, E.D., Reid, L.M. et al. Putative liver progenitor cells: conditions for long-term survival in culture. J Mol Hist 29, 205–217 (1997). https://doi.org/10.1023/A:1026449825608

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