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Expansion Conditions for Early Hepatic Progenitor Cells from Embryonal and Neonatal Rat Livers

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Abstract

Long-term primary cultures were established fromfetal or neonatal livers by using cell suspensionsdepleted of red blood cells and by culturing the cellsin hormonally defined medium containing dimethyl sulfoxide. Two distinct populations of hepaticprogenitor cells were evident in the cultures, based onmorphology, proliferative ability, and liver-specificgene expression. Most colonies consisted of immature hepatic progenitors: small, blastlike cells,weakly expressing alpha-fetoprotein, albumin, andγ-glutamyltranspeptidase, and showing evidence ofproliferation as measured by bromodeoxyuridineincorporation. At the perimeter of these colonies of immaturecells and forming some colonies by themselves were moremature hepatic progenitor cells: larger cells, withincreased cytoplasmic to nuclear ratios, little proliferation, and strongly expressing albumin,alpha-fetoprotein, and γ-glutamyltranspeptidase.The latter two proteins were localized to the bilecanalicular membranes of these cells. Glycogen deposits were present in the mature cells from day 14embryos after eight days of culture. Thus, DMSOtreatment of hepatic parenchymal progenitors provides anovel system for studies of liver development.

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REFERENCES

  1. Shiojiri N, Lemire JM, Fausto N: Cell lineages and oval cell progenitors in rat liver development. Cancer Res 52:2611–2620, 1991

    Google Scholar 

  2. Fre eman AE, Engvall E, Hirata K, Yoshida Y, Kottel RH, Hilborn V, Ruoslahti E: Differentiation of fetal liver cells in vitro. Proc Natl Acad Sci USA 78:3659–3663, 1981

    Google Scholar 

  3. Leffert HL, Paul D: Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine deficient medium. J Cell Physiol 81:113–124, 1972

    Google Scholar 

  4. De Juan C, Benito M, Fabregat I: Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones. J Cell Physiol 152:95–101, 1992

    Google Scholar 

  5. Enat R, Jefferson DM, Ruiz-Opazo N, Gatmaitan Z, Leinwand LM, Reid LM: 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 81:1411–1415, 1984

    Google Scholar 

  6. Re id LM: Defining hormone and matrix requirements for differentiated epithelia. In Me thods in Molecular Biology, Vol 5. JW Pollard, JM Walker (eds). Humana Press, Inc., Clifton, New Jersey, 1990, pp

    Google Scholar 

  7. Michalopuolos GK: Liver regeneration: Molecular me chanisms of growth control. FASEB J 4:176–187, 1990

    Google Scholar 

  8. Rhim JA, Sandgren EP, Degen JL, Palmite r RD, Brinster RL: Replacement of diseased mouse liver by hepatic cell transplantation. Science 263:1149–1152, 1994

    Google Scholar 

  9. Isom H, Georgoff I, Salditt-Georgieff M, Darnell JE, Jr: Persistence of liver-specific messenger RNA in cultured hepatocytes: Different regulatory events for different genes. J Cell Biol 105:2877–2885, 1987

    Google Scholar 

  10. Isom HC, Secott T, Georgoff I, Woodworth C, Mummaw J: Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci USA 82:3252–3256, 1985

    Google Scholar 

  11. Higgins PJ, Borenfreund E: Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dime thylsulfoxide. Biochem Biophys Acta 610:174–180, 1980

    Google Scholar 

  12. De La Vega FM, Mendoza-Figueroa T: Dimethyl sulfoxide enhances lipid synthesis and secretion by long-term cultures of adult hepatocytes. Biochimie 73:621–624, 1991

    Google Scholar 

  13. Baribault H, Marce au N: Dexamethasone and dime thylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocyte s. J Cell Physiol 129:77–84, 1986

    Google Scholar 

  14. Kost DP, Michalopoulos GK: Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture. J Cell Physiol 147:274–280, 1991

    Google Scholar 

  15. McGowan JA: Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide. J Cell Physiol 137:497–504, 1988

    Google Scholar 

  16. Sigal SH, Brill S, Fiorino AS, Re id LM: The liver as a stemcell and lineage system. Am J Physiol 263:G139–G148, 1992

    Google Scholar 

  17. Fausto N: Hepatocyte differentiation and liver progenitor cells. Curr Opin Cell Biol 2:1036–1042, 1990

    Google Scholar 

  18. Germain L, Noe l M, Gourdeau H, Marceau N: Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res 48:368–378, 1988

    Google Scholar 

  19. Sigal S, Brill S, Reid LM, Zvibel I, Gupta S, Hixon D, Faris R, Holst PA: Characterization and enrichment of fetal rat hepatoblasts by immunoadsoption ( “ panning” ) and fluorescence activated cell sorting. Hepatology 19(4):999–1006, 1994

    Google Scholar 

  20. Chessebeuf M, Padieu P: Rat liver epithelial cultures in a serum-free medium: Primary cultures and derived cell lines expressing differentiated functions. In Vitro 20:780–795, 1984

    Google Scholar 

  21. Rutenberg AM, Kim H, Fischbein JW: Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J Histochem Cytochem 17:517–526, 1969

    Google Scholar 

  22. Mitaka T, Mikami M, Sattler GL, Pitot HC, Mochizuki Y: Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Hepatology 16:440–447, 1992

    Google Scholar 

  23. Mitaka T, Norioka K, Nakamura T, Mochizuchi Y: Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes. J Cell Physiol 157:461–468, 1993

    Google Scholar 

  24. Mitaka T, Norioka K, Sattler GL, Pitot HC, Mochizuchi Y: Effect of age on the formation of small-cell colonies in cultures of primary rat hepatocytes. Cancer Res 53:3145–3148, 1993

    Google Scholar 

  25. Mitaka T, Sattler GL, Pitot HC, Mochizuchi Y: Characte ristics of small cell colonies deve loping in primary cultures of adult rat hepatocytes. Virchows Arch B Cell Pathol 62:329–335, 1992

    Google Scholar 

  26. Sawada N, Lee JH, Mochizuki Y, Ishikawa T: Active proliferation of mouse hepatocytes in primary culture under defined conditions as compared to rat hepatocytes. Jpn J Cancer Re s 79:983–988, 1988

    Google Scholar 

  27. Brill S, Zvibe l I, Reid LM: Maturation-dependent changes in the regulation of liver-specific gene expression in embryonal versus adult primary liver cultures. Differentiation 59(2):95–102, 1995

    Google Scholar 

  28. Curran TR Jr, Bahner RI Jr, Oh W, Gruppuso, PA: Mitogenindependent DNA synthesis by fetal rat hepatocytes in primary cultures. Exp Cell Res 209:53–57, 1993

    Google Scholar 

  29. Margolis RN: Regulation of hepatic glycogen metabolism in pre-and postnatal rats. Endocrinology 113:893–902, 1983

    Google Scholar 

  30. Bennett AL, Paulson KE, Miller RE, Darnell JE Jr: Acquisition of antigens characteristic of adult pericentral hepatocytes by differentiating fetal hepatoblasts. J Cell Biol 105:1073–1085, 1987

    Google Scholar 

  31. Earp HS, Lin Q, Blaisde ll J: Dimethyl sulfoxide decreases specific EGF binding. J Cell Biochem 26:221–230, 1984

    Google Scholar 

  32. Tatematsu M, Ho RH, Kaku T, Ekem JK, Farbe r E: Studies on the proliferation and fate of oval cells in the liver of rats treated with 2–ace tylaminofluorene and partial hepatectomy. Am J Pathol 114( 3):418–430, 1984

    Google Scholar 

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Brill, S., Zvibel, I. & Reid, L.M. Expansion Conditions for Early Hepatic Progenitor Cells from Embryonal and Neonatal Rat Livers. Dig Dis Sci 44, 364–371 (1999). https://doi.org/10.1023/A:1026666820422

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