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Differentiation of mouse embryonic stem cells into a defined neuronal lineage

Abstract

Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.

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Figure 1: Schematic representation of the neuronal differentiation procedure.
Figure 2: ES cells differentiate into a homogeneous population of radial glial cells.
Figure 3: Pax-6 is initially expressed by most cells but rapidly disappears.
Figure 4: Neuronal differentiation after 4 d in culture.
Figure 5: Morphology of GFP-positive neurons.
Figure 6: Western blots of extracts of in vitro differentiated neurons prepared at different time points.
Figure 7: Electrophysiological properties of stem cell-derived neurons (see also Supplementary Fig. 1 online).

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. Stavridis, M.P. & Smith, A.G. Neural differentiation of mouse embryonic stem cells. Biochem. Soc. Trans. 31, 45–49 (2003).

    Article  CAS  Google Scholar 

  4. Rathjen, J. & Rathjen, P.D. Mouse ES cells: experimental exploitation of pluripotent differentiation potential. Curr. Opin. Genet. Dev. 11, 587–594 (2001).

    Article  CAS  Google Scholar 

  5. Chung, S. et al. Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. Eur. J. Neurosci. 16, 1829–1838 (2002).

    Article  Google Scholar 

  6. Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).

    Article  CAS  Google Scholar 

  7. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

    Article  CAS  Google Scholar 

  8. Kondo, M. et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 21, 759–806 (2003).

    Article  CAS  Google Scholar 

  9. Lendahl, U., Zimmerman, L.B. & McKay, R.D. CNS stem cells express a new class of intermediate filament protein. Cell 60, 585–595 (1990).

    Article  CAS  Google Scholar 

  10. Li, M., Pevny, L., Lovell-Badge, R. & Smith, A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr. Biol. 8, 971–974 (1998).

    Article  CAS  Google Scholar 

  11. Malatesta, P. et al. Neuronal or glial progeny: regional differences in radial glia fate. Neuron 37, 751–764 (2003).

    Article  CAS  Google Scholar 

  12. Tucker, K.L., Meyer, M. & Barde, Y.A. Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4, 29–37 (2001).

    Article  CAS  Google Scholar 

  13. Bain, G., Kitchens, D., Yao, M., Huettner, J.E. & Gottlieb, D.I. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342–357 (1995).

    Article  CAS  Google Scholar 

  14. Li, M. Lineage selection for generation and amplification of neural precursor cells. in Methods in Molecular Biology: Embryonic Stem Cells: Methods and Protocols 185, 205–215 (Humana Press, Totowa, New Jersey, USA, 2002).

    Google Scholar 

  15. Brewer, G.J. & Cotman, C.W. Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res. 494, 65–74 (1989).

    Article  CAS  Google Scholar 

  16. Hartfuss, E., Galli, R., Heins, N. & Götz, M. Characterization of CNS precursor subtypes and radial glia. Dev. Biol. 229, 15–30 (2001).

    Article  CAS  Google Scholar 

  17. Misson, J.P., Edwards, M.A., Yamamoto, M. & Caviness, V.S. Jr. Identification of radial glial cells within the developing murine central nervous system: studies based upon a new immunohistochemical marker. Brain Res. Dev. Brain Res. 44, 95–108 (1988).

    Article  CAS  Google Scholar 

  18. Feng, L., Hatten, M.E. & Heintz, N. Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron 12, 895–908 (1994).

    Article  CAS  Google Scholar 

  19. Götz, M., Stoykova, A. & Gruss, P. Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21, 1031–1044 (1998).

    Article  Google Scholar 

  20. Banker, G.A. & Cowan, W.M. Rat hippocampal neurons in dispersed cell culture. Brain Res. 126, 397–442 (1977).

    Article  CAS  Google Scholar 

  21. Fremeau, R.T., Jr et al. The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31, 247–260 (2001).

    Article  CAS  Google Scholar 

  22. Klein, R., Martin-Zanca, D., Barbacid, M. & Parada, L.F. Expression of the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system. Development 109, 845–850 (1990).

    CAS  PubMed  Google Scholar 

  23. Bothwell, M. Functional interactions of neurotrophins and neurotrophin receptors. Annu. Rev. Neurosci. 18, 223–253 (1995).

    Article  CAS  Google Scholar 

  24. Trapp, B.D. & Hauer, P.E. Amyloid precursor protein is enriched in radial glia: implications for neuronal development. J. Neurosci. Res. 37, 538–550 (1994).

    Article  CAS  Google Scholar 

  25. Vicario-Abejón, C., Collin, C., McKay, R.D. & Segal, M. Neurotrophins induce formation of functional excitatory and inhibitory synapses between cultured hippocampal neurons. J. Neurosci. 18, 7256–7271 (1998).

    Article  Google Scholar 

  26. Song, H.J., Stevens, C.F. & Gage, F.H. Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat. Neurosci. 5, 438–445 (2002).

    Article  CAS  Google Scholar 

  27. Fraichard, A. et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 108, 3181–3188 (1995).

    CAS  PubMed  Google Scholar 

  28. Strübing, C. et al. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech. Dev. 53, 275–287 (1995).

    Article  Google Scholar 

  29. Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M. & McKay, R.D. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59, 89–102 (1996).

    Article  CAS  Google Scholar 

  30. Renoncourt, Y., Carroll, P., Filippi, P., Arce, V. & Alonso, S. Neurons derived in vitro from ES cells express homeoproteins characteristic of motoneurons and interneurons. Mech. Dev. 79, 185–197 (1998).

    Article  CAS  Google Scholar 

  31. Rathjen, J. et al. Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development 129, 2649–2661 (2002).

    CAS  PubMed  Google Scholar 

  32. Abe, Y. et al. Analysis of neurons created from wild-type and Alzheimer's mutation knock-in embryonic stem cells by a highly efficient differentiation protocol. J. Neurosci. 23, 8513–8525 (2003).

    Article  CAS  Google Scholar 

  33. Barberi, T. et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat. Biotechnol. 21, 1200–1207 (2003).

    Article  CAS  Google Scholar 

  34. Jones-Villeneuve, E.M., McBurney, M.W., Rogers, K.A. & Kalnins, V.I. Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells. J. Cell Biol. 94, 253–262 (1982).

    Article  CAS  Google Scholar 

  35. Bain, G., Ray, W.J., Yao, M. & Gottlieb, D.I. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem. Biophys. Res. Commun. 223, 691–694 (1996).

    Article  CAS  Google Scholar 

  36. Murray, P. & Edgar, D. The topographical regulation of embryonic stem cell differentiation. Proc. R. Soc. Meeting Rev. (in the press).

  37. Liour, S.S. & Yu, R.K. Differentiation of radial glia-like cells from embryonic stem cells. Glia 42, 109–117 (2003).

    Article  Google Scholar 

  38. Aubert, J., Dunstan, H., Chambers, I. & Smith, A. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat. Biotechnol. 20, 1240–1245 (2002).

    Article  CAS  Google Scholar 

  39. Kim, A.S., Lowenstein, D.H. & Pleasure, S.J. Wnt receptors and Wnt inhibitors are expressed in gradients in the developing telencephalon. Mech. Dev. 103, 167–172 (2001).

    Article  CAS  Google Scholar 

  40. Gajovic, S., St-Onge, L., Yokota, Y. & Gruss, P. Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells. Differentiation 62, 187–192 (1997).

    CAS  PubMed  Google Scholar 

  41. Diez del Corral, R. et al. Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65–79 (2003).

    Article  CAS  Google Scholar 

  42. Novitch, B.G., Wichterle, H., Jessell, T.M. & Sockanathan, S. A requirement for retinoic acid-mediated transcriptional activation in ventral neural patterning and motor neuron specification. Neuron 40, 81–95 (2003).

    Article  CAS  Google Scholar 

  43. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–200 (1991).

    Article  CAS  Google Scholar 

  44. Tucker, K.L., Wang, Y., Dausman, J. & Jaenisch, R.A. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 18, 3745–3746 (1997).

    Article  Google Scholar 

  45. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Smith and M. Li for allowing M.B. to learn techniques of ES cell differentiation in their laboratory and for valuable suggestions, and D. Gerosa for technical assistance.

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Correspondence to Yves-Alain Barde.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Detailed electrophysiological characteristics of stem cell-derived neurons. (a) Method to calculate cell properties from Table 2. Tau was calculated as the time needed to reach 63% of the membrane voltage at the end of the hyperpolarizing pulse. (b) Increase of spontaneous activity by application of 50 μM bicuculline (current-clamp recording) clearly shows the existence of GABAergic input to the recorded cell that was 20-d old. (c) Cell response to a 2 s hyper- and depolarizing current pulse. (Left cell 14 DIV, right cell 20 DIV). From 20 cells tested 13 could follow the depolarization pulse with action potentials during the whole time, whereas seven cells only could follow approx. one-third of the time. (d) To further distinguish between EPSCs and IPSCs the cell (20 DIV) was clamped to -30 mV which is above the reversal potential of IPSCs. Left column: holding potential -60 mV, right column: holding potential -30 mV. In normal ACSF, EPSCs and IPSCs are present at -30 mV (top row). During the application of NBQX/AP5 only IPSCs could be detected (second row), whereas all responses could be blocked by further application of bicuculline (third row). The bottom row shows responses after washout of the drugs. (GIF 207 kb)

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Bibel, M., Richter, J., Schrenk, K. et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci 7, 1003–1009 (2004). https://doi.org/10.1038/nn1301

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