Skip to main content
NCBI home page
The American Journal of Pathology logoLink to The American Journal of Pathology
. 1996 Nov;149(5):1593–1605.

Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells.

C Tateno 1, K Yoshizato 1
PMCID: PMC1865270  PMID: 8909249

Abstract

A cell fraction containing small hepatocytes and nonparenchymal cells was isolated from the adult rat liver and was cultured in the presence of vitamin C. epidermal growth factor, nicotinamide, and dimethylsulfoxide. All of the small hepatocytes that had attached to a dish expressed hepatocytic phenotypes such as albumin, transferrin, and cytokeratin (CK)8 and CK18 but not biliary markers such as BD1, CK7, and CK19. Single hepatocytes started to proliferate and grew into colonies. The growth potential was variable among the cells, the highest case being that a single cell produced a colony containing over 100 cells in 10 days. The hepatocytes in the colony developed through a proliferation phase and then a differentiation phase. They showed very high bromodeoxyuridine labeling indexes during the first 7 days (proliferation phase), which gradually decreased thereafter. Phenotypic alterations took place at 7 to 10 days, and some hepatocytes started to express mature hepatocyte markers and biliary markers (differentiation phase). The presence of cells that coexpress albumin and biliary markers (CK7 and CK19) was demonstrated by double immunocytochemistry. In addition, cells were identified that ceased to express albumin and in turn were positive for CK19 or CK7. Therefore, the colony was considered to contain liver progenitor-like cells that can differentiate during culture into cells expressing phenotypes of mature hepatocytes or biliary epithelial cells.

Full text

PDF
1593

Images in this article

1597Figure 2
on p.1597
1600Figure 6
on p.1600
1602Figure 7
on p.1602

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bisgaard H. C., Nagy P., Ton P. T., Hu Z., Thorgeirsson S. S. Modulation of keratin 14 and alpha-fetoprotein expression during hepatic oval cell proliferation and liver regeneration. J Cell Physiol. 1994 Jun;159(3):475–484. doi: 10.1002/jcp.1041590312. [DOI] [PubMed] [Google Scholar]
  2. Grisham J. W., Coleman W. B., Smith G. J. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med. 1993 Dec;204(3):270–279. doi: 10.3181/00379727-204-43663. [DOI] [PubMed] [Google Scholar]
  3. Guillouzo A., Delers F., Clement B., Bernard N., Engler R. Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium. Biochem Biophys Res Commun. 1984 Apr 30;120(2):311–317. doi: 10.1016/0006-291x(84)91255-5. [DOI] [PubMed] [Google Scholar]
  4. Loréal O., Levavasseur F., Fromaget C., Gros D., Guillouzo A., Clément B. Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro. Am J Pathol. 1993 Aug;143(2):538–544. [PMC free article] [PubMed] [Google Scholar]
  5. Mitaka T., Kojima T., Mizuguchi T., Mochizuki Y. Growth and maturation of small hepatocytes isolated from adult rat liver. Biochem Biophys Res Commun. 1995 Sep 14;214(2):310–317. doi: 10.1006/bbrc.1995.2289. [DOI] [PubMed] [Google Scholar]
  6. Mitaka T., Sattler C. A., Sattler G. L., Sargent L. M., Pitot H. C. Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology. 1991 Jan;13(1):21–30. [PubMed] [Google Scholar]
  7. Mito M., Ebata H., Kusano M., Onishi T., Saito T., Sakamoto S. Morphology and function of isolated hepatocytes transplanted into rat spleen. Transplantation. 1979 Dec;28(6):499–505. doi: 10.1097/00007890-197912000-00013. [DOI] [PubMed] [Google Scholar]
  8. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  9. Mossin L., Blankson H., Huitfeldt H., Seglen P. O. Ploidy-dependent growth and binucleation in cultured rat hepatocytes. Exp Cell Res. 1994 Oct;214(2):551–560. doi: 10.1006/excr.1994.1293. [DOI] [PubMed] [Google Scholar]
  10. Rhim J. A., Sandgren E. P., Degen J. L., Palmiter R. D., Brinster R. L. Replacement of diseased mouse liver by hepatic cell transplantation. Science. 1994 Feb 25;263(5150):1149–1152. doi: 10.1126/science.8108734. [DOI] [PubMed] [Google Scholar]
  11. Sandgren E. P., Palmiter R. D., Heckel J. L., Daugherty C. C., Brinster R. L., Degen J. L. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell. 1991 Jul 26;66(2):245–256. doi: 10.1016/0092-8674(91)90615-6. [DOI] [PubMed] [Google Scholar]
  12. Sawada N., Lee G. H., Mochizuki Y., Ishikawa T. Active proliferation of mouse hepatocytes in primary culture under defined conditions as compared to rat hepatocytes. Jpn J Cancer Res. 1988 Sep;79(9):983–988. doi: 10.1111/j.1349-7006.1988.tb00064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  14. Sell S. Is there a liver stem cell? Cancer Res. 1990 Jul 1;50(13):3811–3815. [PubMed] [Google Scholar]
  15. Sell S., Leffert H. L. An evaluation of cellular lineages in the pathogenesis of experimental hepatocellular carcinoma. Hepatology. 1982 Jan-Feb;2(1):77–86. doi: 10.1002/hep.1840020113. [DOI] [PubMed] [Google Scholar]
  16. Senoo H., Hata R. Extracellular matrix regulates and L-ascorbic acid 2-phosphate further modulates morphology, proliferation, and collagen synthesis of perisinusoidal stellate cells. Biochem Biophys Res Commun. 1994 Apr 29;200(2):999–1006. doi: 10.1006/bbrc.1994.1549. [DOI] [PubMed] [Google Scholar]
  17. Shiojiri N., Lemire J. M., Fausto N. Cell lineages and oval cell progenitors in rat liver development. Cancer Res. 1991 May 15;51(10):2611–2620. [PubMed] [Google Scholar]
  18. Sirica A. E., Richards W., Tsukada Y., Sattler C. A., Pitot H. C. Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc Natl Acad Sci U S A. 1979 Jan;76(1):283–287. doi: 10.1073/pnas.76.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tateno C., Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes. Am J Pathol. 1996 Feb;148(2):383–392. [PMC free article] [PubMed] [Google Scholar]
  20. Van Eyken P., Sciot R., Desmet V. J. A cytokeratin immunohistochemical study of alcoholic liver disease: evidence that hepatocytes can express 'bile duct-type' cytokeratins. Histopathology. 1988 Dec;13(6):605–617. doi: 10.1111/j.1365-2559.1988.tb02092.x. [DOI] [PubMed] [Google Scholar]
  21. Van Eyken P., Sciot R., Desmet V. J. A cytokeratin immunohistochemical study of cholestatic liver disease: evidence that hepatocytes can express 'bile duct-type' cytokeratins. Histopathology. 1989 Aug;15(2):125–135. doi: 10.1111/j.1365-2559.1989.tb03060.x. [DOI] [PubMed] [Google Scholar]
  22. Wu J. C., Merlino G., Cveklova K., Mosinger B., Jr, Fausto N. Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1. Cancer Res. 1994 Nov 15;54(22):5964–5973. [PubMed] [Google Scholar]
  23. Yang L., Faris R. A., Hixson D. C. Characterization of a mature bile duct antigen expressed on a subpopulation of biliary ductular cells but absent from oval cells. Hepatology. 1993 Aug;18(2):357–366. [PubMed] [Google Scholar]
  24. Zhang M., Thorgeirsson S. S. Modulation of connexins during differentiation of oval cells into hepatocytes. Exp Cell Res. 1994 Jul;213(1):37–42. doi: 10.1006/excr.1994.1170. [DOI] [PubMed] [Google Scholar]

Articles from The American Journal of Pathology are provided here courtesy of American Society for Investigative Pathology

RESOURCES