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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).
Lee, H. et al. Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25, 1931–1939 (2007).
Sasai, Y. et al. Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779–790 (1994).
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).
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).
Valenzuela, D.M. et al. Identification of mammalian noggin and its expression in the adult nervous system. J. Neurosci. 15, 6077–6084 (1995).
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).
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).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Callaerts, P., Halder, G. & Gehring, W.J. PAX-6 in development and evolution. Annu. Rev. Neurosci. 20, 483–532 (1997).
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).
Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).
D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).
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).
Munoz-Sanjuan, I. & Brivanlou, A.H. Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3, 271–280 (2002).
Tesar, P.J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007).
Li, X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005).
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).
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).
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).
Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).
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.).
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
Circulation Research (2021)
Journal of Pharmacology and Experimental Therapeutics (2021)
Cell Stem Cell (2021)
Experimental Neurology (2021)
Neurochemical Research (2021)