Advertisement

Neural Stem Sphere Method

Induction of Neural Stem Cells and Neurons by Astrocyte-Derived Factors in Embryonic Stem Cells In Vitro
  • Takashi Nakayama
  • Nobuo Inoue
Part of the Methods in Molecular Biology™ book series (MIMB, volume 330)

Summary

We have developed a novel method to produce large numbers of neural stem cells and neurons directly from embryonic stem (ES) cells. The method is composed of three culture stages. In the first stage, floating ES cell colonies are cultured in astrocyte-conditioned medium to directly differentiate neural stem cells from ES cells, resulting in neural stem spheres. In the second stage, neural stem spheres are cultured as adhesive cells to produce neural stem cells by cell migration. In the third stage, neural stem cells are cultured as monolayers, which proliferate in the presence of fibroblast growth factor 2 and epidermal growth factor and differentiate into neurons in astrocyte-conditioned medium.

Key Words

Astrocyte-conditioned medium embryonic stem cells neural stem cells neural stem sphere 

References

  1. 1.
    Okabe S., Forsberg-Nilsson K., Spiro A. C., Segal M., and McKay R. D. (1996) Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59, 89–102.CrossRefPubMedGoogle Scholar
  2. 2.
    Kawasaki H., Mizuseki K., Nishikawa S., et al. (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40.CrossRefPubMedGoogle Scholar
  3. 3.
    Nakayama T., Momoki-Soga T., and Inoue N. (2003) Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons. Neurosci. Res. 46, 241–249.PubMedGoogle Scholar
  4. 4.
    Nakayama T., Momoki-Soga T., Yamaguchi K., and Inoue N. (2004) Efficient production of neural stem cells and neurons from embryonic stem cells. Neuroreport 15, 487–491.CrossRefPubMedGoogle Scholar
  5. 5.
    Inoue N., Matsui H., Tsukui H., and Hatanaka H. (1988) The appearance of a highly digitalis-sensitive isoform of Na +,K +-ATPase during maturation in vitro of primary cultured rat cerebral neurons. J. Biochem. 104, 349–354.PubMedGoogle Scholar
  6. 6.
    Noble M. and Mayer-Pörschel M. (1998) Culture of astrocytes, oligodendrocytes, and 0-2A progenitor cells, in Culturing Nerve Cells, 2nd ed. (Banker G., and Goslin K., eds)., MIT Press Cambridge, MA, pp. 499–543.Google Scholar
  7. 7.
    Barberi T., Klivenyi P., Calingasan N. Y., et al. (2003) Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat. Biotechnol. 21, 1200–1207.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Takashi Nakayama
    • 1
  • Nobuo Inoue
    • 2
  1. 1.Department of BiochemistryYokohama City University School of MedicineYokohamaJapan
  2. 2.Laboratory of Regenerative Neurosciences, Faculty of Health SciencesTokyo MetropolitanTokyoJapan

Personalised recommendations