Transgenic Research

, Volume 3, Issue 3, pp 152–158 | Cite as

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

  • Eckhard Wolf
  • Rainer Kramer
  • Irina Polejaeva
  • Hans Thoenen
  • Gottfried Brem


The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.


CNTF LIF ES cells differentiation proliferation chimaera 


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  1. Bradley, A., Evans, M., Kaufman, M.H. and Robertson, E. (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.Nature 309, 255–6.CrossRefPubMedGoogle Scholar
  2. Capecchi, M.R. (1989) The new mouse genetics: altering the genome by gene targeting.Trends Genet. 5, 70–6.CrossRefPubMedGoogle Scholar
  3. Chang, J.M. and Gough, N.M. (1992) The diverse activities of leukaemia inhibitory factor: from embryonic stem cells to platelets.Focus 14, 6–9.Google Scholar
  4. Coon, H.G. (1968) Clonal culture of differentiated rat liver cells.J. Cell Biol. 39, 29a.Google Scholar
  5. Davis, S., Aldrich, T.H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N.Y. and Yancopoulos, G.D. (1993a) LIFRβ and gp130 as heterodimerizing signal transducers of the tripartite CNTF receptor.Science 260, 1805–8.PubMedGoogle Scholar
  6. Davis, S., Aldrich, T.H., Ip, N.Y., Stahl, N., Scherer, S., Farruggella, T., DiStefano, P.S., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S. and Yancopoulos, G.D. (1993b) Released form of CNTF receptor α component as soluble mediator of CNTF responses.Science 259, 1736–9.PubMedGoogle Scholar
  7. Doetschman, T., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R. (1985) Thein vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.J. Embryol. Exp. Morph. 87, 27–45.PubMedGoogle Scholar
  8. Evans, M.J. and Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos.Nature 292, 154–6.CrossRefPubMedGoogle Scholar
  9. Gearing, D.P., Nicola, N.A., Metcalf, D., Foote, S., Willson, T.A., Gough, N.M. and Williams, R.L. (1989) Production of leukaemia inhibitory factor inEscherichia coli by a novel procedure and its use in maintaining embryonic stem cells in culture.Bio/Technology 7, 1157–61.Google Scholar
  10. Gearing, D.P., Comeau, M.R., Friend, D.J., Gimpel, S.D., Thut, C.J., McGourty, J., Brasher, K.K., King, J.A., Gillis, S., Mosley, B., Ziegler, S.F. and Cosman, D. (1992) The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor.Science 255, 1434–7.PubMedGoogle Scholar
  11. Hilton, D.J. and Gough, N.M. (1991) Leukaemia inhibitory factor: a biological perspective.J. Cell. Biochem. 46, 21–6.CrossRefPubMedGoogle Scholar
  12. Hooper, M.L. (1992)Embryonal Stem Cells: Introducing Planned Changes into the Animal Germline. Chur, Switzerland: Harwood Academic Publishers GmbH.Google Scholar
  13. Ip, N.Y., Nye, S.H., Boulton, T.G., Davis, S., Taga, T., Li, Y., Birren, S.J., Yasukawa, K., Kishimoto, T., Anderson, D.J., Stahl, N. and Yancopoulos, G.D. (1992) CNTF and LIF act on neuronal cells via shared signaling pathways that involve the IL-6 signal transducing receptor component gp130.Cell 69, 1121–32.CrossRefPubMedGoogle Scholar
  14. Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S. and Yancopoulos, G.D. (1993) The α component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development.Neuron 10, 89–102.CrossRefPubMedGoogle Scholar
  15. Joyner, A.L., Herrup, K., Auerbach, B.A., Davis, C.A. and Rossant, J. (1991) Subtle cerebella phenotype in mice homozygous for a targeted deletion in theEn-2 homeobox.Science 251, 1239–43.PubMedGoogle Scholar
  16. Le Mouellic, H., Lallemand, Y. and Brûlet, P. (1992) Homeosis in the mouse induced by a null mutation in theHox-3.1 gene.Cell 69, 251–64.CrossRefPubMedGoogle Scholar
  17. Lufkin, T., Dierich, A., Lemeur, M., Mark, M. and Chambon, P. (1991) Disruption of theHox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression.Cell 66, 1105–19.CrossRefPubMedGoogle Scholar
  18. Marquardt, H., Todaro, G.J., Henderson, L.E. and Oroszlan, S. (1981) Purification and primary structure of a polypeptide with multiplication stimulating activity from rat liver cell cultures.J. Biol. Chem. 256, 6859–65.PubMedGoogle Scholar
  19. Martin, G.R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc. Natl Acad. Sci. USA 78, 7634–8.PubMedGoogle Scholar
  20. Massagué, J., Kelly, B. and Mottola, C. (1985) Stimulation by insulin-like growth factors is required for cellular transformation by type β transforming growth factor.J. Biol. Chem. 260, 4551–4.PubMedGoogle Scholar
  21. Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G. and Thoenen, H. (1993) Disruption of the CNTF gene results in motor neural degeneration.Nature. 365, 27–32.CrossRefPubMedGoogle Scholar
  22. Metcalf, D. (1991) The leukaemia inhibitory factor (LIF).Int. J. Cell Cloning 9, 95–108.PubMedGoogle Scholar
  23. Mummery, C.L., Feyen, A., Freund, E. and Shen, S. (1990) Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines.Cell Differ. Dev. 30, 195–206.CrossRefPubMedGoogle Scholar
  24. Nagy, A., Gócza, E., Diaz, E.M., Prideaux, V.R., Iványi, E., Markkula, M. and Rossant, J. (1990) Embryonic stem cells alone are able to support fetal development in the mouse.Development 110, 815–21.PubMedGoogle Scholar
  25. Nichols, J., Evans, E.P. and Smith, A.G. (1990) Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity.Development 110, 1341–8.PubMedGoogle Scholar
  26. Pease, S. and Williams, R.L. (1990) Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukaemia inhibitory factor.Exp. Cell Res. 190, 209–11.CrossRefPubMedGoogle Scholar
  27. Pease, S., Braghetta, P., Gearing, D., Grail, D. and Williams, R.L. (1990) Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukaemia inhibitory factor (LIF).Dev. Biol. 141, 344–52.CrossRefPubMedGoogle Scholar
  28. Quinn, P., Barros, C. and Whittingham, D.G. (1982) Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa.J. Reprod. Fertil. 66, 161–8.PubMedGoogle Scholar
  29. Rossant, J. and Hopkins, N. (1992) Of fin and fur: mutational analysis of vertebrate embryonic development.Genes Devel. 6, 1–13.PubMedGoogle Scholar
  30. Sendtner, M., Arakawa, Y., Stöckli, K.A., Kreutzberg, G.W. and Thoenen, H. (1991) Effect of ciliary neurotrophic factor (CNTF) on motoneuron survival.J. Cell Sci. Suppl. 15, 103–9.PubMedGoogle Scholar
  31. Smith, A.G. and Hooper, M.L. (1987) Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.Dev. Biol. 121, 1–9.CrossRefPubMedGoogle Scholar
  32. Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J., Stahl, M. and Rogers, D. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.Nature 336, 688–90.CrossRefPubMedGoogle Scholar
  33. Solter, D. and Knowles, B.B. (1978) Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1).Proc. Natl Acad. Sci. USA 75, 5565–9.PubMedGoogle Scholar
  34. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A. and Gough, N.M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.Nature 336, 684–7.CrossRefPubMedGoogle Scholar
  35. Wolf, E., Masu, Y., Thoenen, H. and Brem, G. (1993) Germline transmission of murine embryonic stem (ES) cells after targeted inactivation of one allele of the CNTF gene.Theriogenology 39, 341.CrossRefGoogle Scholar
  36. Wood, S.A., Pascoe, W.S., Schmidt, C., Kemler, R., Evans, M.J. and Allen, N.D. (1993) Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.Proc. Natl Acad. Sci. USA 90, 4582–5.PubMedGoogle Scholar
  37. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. and Jaenisch, R. (1989) Germ-line transmission of a disrupted β2-microglobulin gene produced by homologous recombination in embryonic stem cells.Nature 342, 435–8.CrossRefPubMedGoogle Scholar

Copyright information

© Chapman & Hall 1994

Authors and Affiliations

  • Eckhard Wolf
    • 1
  • Rainer Kramer
    • 2
  • Irina Polejaeva
    • 1
  • Hans Thoenen
    • 2
  • Gottfried Brem
    • 1
  1. 1.Lehrstuhl für Molekulare TierzuchtLudwig-Maximilians-UniversitätMünchenGermany
  2. 2.Abteilung NeurochemieMax-Planck-Institut für PsychiatrieMartinsriedGermany

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