Retinoids pp 75-83 | Cite as

Feeder-Independent Culture of Mouse Embryonic Stem Cells Using Vitamin A/Retinol

  • Jaspal S. Khillan
  • Liguo Chen
Part of the Methods in Molecular Biology book series (MIMB, volume 652)


Embryonic stem (ES) cells derived from the inner cell mass of a mammalian blastocyst represent unlimited source of all types of cells for regenerative medicine and for drug discovery. Mouse and human ES cells require mouse embryonic fibroblast feeder cells to maintain their undifferentiated state which involve additional time-consuming and labor-intensive steps. Recently we reported a novel function of retinol, the alcohol form of vitamin A, in preventing the differentiation of mouse ES cells. Retinol/vitamin A induces the overexpression of Nanog, a key transcription factor that is important for maintaining the pluripotency of mouse and human ES cells. Further, retinol/vitamin A also supports feeder-independent culture of ES cells in long-term cultures. The cells continue to maintain the expression of pluripotent cell-specific markers such as Nanog, Oct4, and Sox2 and form chimeric animals after injection into blastocysts. In this chapter, we describe feeder-independent cultures of mouse ES cells in the medium supplemented with retinol. The ES cells are cultured over plates coated with gelatin in ES medium with leukemia inhibitory factor (LIF) which is supplemented with 0.5 μM retinol/vitamin A. The cells are passaged every 3–5 days by trypsinization. The pluripotency of the cells is tested by different undifferentiated ES cell-specific markers.

Key words

Self-renewal of ES cells feeder-independent cultures vitamin A/retinol Nanog regulation Adh1 Adh4 RALDH2 


  1. 1.
    Martin, G. (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, 7634–7638.PubMedCrossRefGoogle Scholar
  2. 2.
    Evans, M.J., Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.PubMedCrossRefGoogle Scholar
  3. 3.
    Boiani, M., Scholer, H.R. (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell. Biol. 6, 872–881.Google Scholar
  4. 4.
    Dahéron, L., Opitz, S.L., Zaehres, H., Lensch, M.W., Andrews, P.W., Itskovitz-Eldor, J., Daley, G.Q. (2004) LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 22, 770–778.PubMedCrossRefGoogle Scholar
  5. 5.
    Robertson, E.J. (1987) Embryo derived cell lines. In: Teratocarcinoma and Embryonic Stem Cells: A Practical Approach, IRL Press, Oxford, pp. 71–112.Google Scholar
  6. 6.
    Ludwig, T., Thomson, J.A. (2007) Defined, feeder-independent medium for human embryonic stem cell culture. Curr. Protoc. Stem Cell Biol. Chapter 1, Unit 1C.2.Google Scholar
  7. 7.
    Chen, LG., Yang, M., Dawes, J., Khillan, J.S. (2007) Suppression of ES cell differentiation by retinol (vitamin A) via the over expression of Nanog. Differentiation 75, 682–693.PubMedCrossRefGoogle Scholar
  8. 8.
    Chen, L., Khillan, J.S. (2008) Promotion of feeder independent self-renewal of embryonic stem cells by retinol (vitamin A). Stem Cells 26, 1858–1864.PubMedCrossRefGoogle Scholar
  9. 9.
    Clagett-Dame, M., De Luca, H.F. (2002) The role of vitamin A in mammalian reproduction and embryonic development. Annu. Rev. Nutr. 22, 347–381.PubMedCrossRefGoogle Scholar
  10. 10.
    Mark, M., Ghyselinck, N.B., Chambon, P. (2006) Function of retinoid nuclear receptors: Lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis. Annu. Rev. Pharmacol. Toxicol. 46, 451–480.PubMedCrossRefGoogle Scholar
  11. 11.
    Kawaguchi, R., Yu, J., Honda, J., Hu, J., Whitelegge, J., Ping, P., et al. (2007) Membrane receptor for retinol binding protein mediates cellular uptake of vitamin A. Science 315, 820–825.PubMedCrossRefGoogle Scholar
  12. 12.
    Lane, M.A., Xu, J., Wilen, E.W., Sylvester, R., Derguini, F., Gudas, L.J. (2008) LIF removal increases CRABPI and CRABPII transcripts in embryonic stem cells cultured in retinol or 4-oxoretinol. Mol. Cell Endocrinol. 280, 63–67.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Jaspal S. Khillan
    • 1
  • Liguo Chen
    • 1
  1. 1.Department of Microbiology and Molecular GeneticsUniversity of PittsburghPittsburghUSA

Personalised recommendations