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Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Abstract

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.

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Figure 1
Figure 2: Analysis of pluripotency of embryonic stem (ES) cells grown in ESN2 medium.

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References

  1. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  2. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  3. Bradley, A., Evans, M., Kaufman, M.H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).

    Article  CAS  Google Scholar 

  4. Shimizukawa, R. et al. Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously. Genesis 42, 47–52 (2005).

    Article  CAS  Google Scholar 

  5. Ying, Q.L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003).

    Article  CAS  Google Scholar 

  6. Fok, E.Y. & Zandstra, P.W. Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 23, 1333–1342 (2005).

    Article  CAS  Google Scholar 

  7. Hayashi, Y. et al. Integrins regulate mouse embryonic stem cell self-renewal. Stem Cells 25, 3005–3015 (2007).

    Article  CAS  Google Scholar 

  8. zur Nieden, N.I., Cormier, J.T., Rancourt, D.E. & Kallos, M.S. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors. J. Biotechnol. 129, 421–432 (2007).

    Article  CAS  Google Scholar 

  9. Bottenstein, J.E. & Sato, G.H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. USA

  10. Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    Article  CAS  Google Scholar 

  11. Andäng, M. et al. Histone H2AX-dependent GABAA receptor regulation of stem cell proliferation. Nature 451, 460–464 (2008).

    Article  Google Scholar 

  12. Moliner, A., Ibanez, C.F., Ernfors, P. & Andäng, M. Mouse embryonic stem cell-derived spheres with distinct neurogenic potentials. Stem Cells Dev. (in the press).

  13. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Swedish Research Council, the Swedish foundation for strategic research, CEDB and DBRM grants (to P.E., C.F.I.), Marcus and Marianne Wallenberg Foundation (to C.F.I.), the Swedish Cancer Foundation, the Swedish Brain Foundation and the Bertil Hållsten Research Foundation (to P.E.). M.A. was supported by grants from the Karolinska Institutet and the Swedish Brain Foundation.

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Correspondence to Michael Andäng or Patrik Ernfors.

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Andäng, M., Moliner, A., Doege, C. et al. Optimized mouse ES cell culture system by suspension growth in a fully defined medium. Nat Protoc 3, 1013–1017 (2008). https://doi.org/10.1038/nprot.2008.65

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