Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

A Corrigendum to this article was published on 01 May 2009

This article has been updated

Abstract

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5+ epiblast-like stage followed by PAX6+ neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Dual-SMAD inhibition allows for highly efficient feeder-free neural induction in adherent cultures in 7 d.
Figure 2: Neuralization of hES cells by dual-SMAD inhibition permits a pre-rosette, neural stem cell with dopaminergic and motoneuronal potential.
Figure 3: IPS cells can be differentiated to neural tissue using dual-SMAD inhibition and are patternable to dopaminergic neurons and motoneurons.
Figure 4: Model of proposed mechanisms that contribute to the action of Noggin and SB431542.

Change history

  • 16 March 2009

    In the version of this article initially published, the unit (nM) for the amount of TGF-b inhibitor (Tocris) reported in the Methods section “Neural induction” was incorrect. The correct unit is μM. The error has been corrected in the HTML and PDF versions of the article.

References

  1. 1

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Lee, H. et al. Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25, 1931–1939 (2007).

    CAS  Article  Google Scholar 

  4. 4

    Sasai, Y. et al. Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779–790 (1994).

    CAS  Article  Google Scholar 

  5. 5

    Hemmati-Brivanlou, A., Kelly, O.G. & Melton, D.A. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77, 283–295 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Smith, W.C. & Harland, R.M. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 829–840 (1992).

    CAS  Article  Google Scholar 

  7. 7

    Valenzuela, D.M. et al. Identification of mammalian noggin and its expression in the adult nervous system. J. Neurosci. 15, 6077–6084 (1995).

    CAS  Article  Google Scholar 

  8. 8

    Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22, 152–165 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Smith, J.R. et al. Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev. Biol. 313, 107–117 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Callaerts, P., Halder, G. & Gehring, W.J. PAX-6 in development and evolution. Annu. Rev. Neurosci. 20, 483–532 (1997).

    CAS  Article  Google Scholar 

  12. 12

    Xu, R.H. et al. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3, 196–206 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).

    CAS  Article  Google Scholar 

  14. 14

    D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Laflamme, M.A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Munoz-Sanjuan, I. & Brivanlou, A.H. Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3, 271–280 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Li, X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005).

    Article  Google Scholar 

  19. 19

    Suter, D.M., Tirefort, D., Julien, S. & Krause, K.H.A. Sox1 to Pax6 switch drives neuroectoderm to radial glia progression during differentiation of mouse embryonic stem cells. Stem Cells 27, 49–58 (2008).

    Article  Google Scholar 

  20. 20

    Tomishima, M.J., Hadjantonakis, A.K., Gong, S. & Studer, L. Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library. Stem Cells 25, 39–45 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Placantonakis, D.G. et al. Bac transgenesis in human Es cells as a novel tool to define the human neural lineage. Stem Cells published online, doi:10.1634/stemcells.2008-0884 (11 December 2008).

  22. 22

    Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to F. Vaccarino for providing Otx2 antibody and E. Lai for BF1 antibody. This work was supported in part by the Starr Foundation, NINDS grant 1R01NS052671, the Starr Stem Scholar fellowship (S.M.C.) and the a New York Stem Cell Foundation fellowship (C.A.F.).

Author information

Affiliations

Authors

Contributions

S.M.C. and L.S. designed the study. E.P.P., M.T., L.S. and M.S. designed and generated the hiPS clones. S.M.C. and L.S. analyzed the data and wrote the manuscript. S.M.C. and C.A.F. performed the experiments.

Corresponding authors

Correspondence to Stuart M Chambers or Lorenz Studer.

Supplementary information

Supplementary Text and Figures

Figures 1–6 (PDF 2665 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chambers, S., Fasano, C., Papapetrou, E. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275–280 (2009). https://doi.org/10.1038/nbt.1529

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing