Abstract
An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube–like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro–generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.
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References
Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).
Briscoe, J. & Ericson, J. Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11, 43–49 (2001).
Bally-Cuif, L. & Hammerschmidt, M. Induction and patterning of neuronal development, and its connection to cell cycle control. Curr. Opin. Neurobiol. 13, 16–25 (2003).
Vasiliauskas, D. & Stern, C.D. Patterning the embryonic axis: FGF signaling and how vertebrate embryos measure time. Cell 106, 133–136 (2001).
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).
Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).
Tropepe, V. et al. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30, 65–78 (2001).
Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).
Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).
Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. & McKay, R.D. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675–679 (2000).
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).
Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).
Pevny, L.H., Sockanathan, S., Placzek, M. & Lovell-Badge, R. A role for SOX1 in neural determination. Development 125, 1967–1978 (1998).
Stern, C.D. Initial patterning of the central nervous system: how many organizers? Nat. Rev. Neurosci. 2, 92–98 (2001).
Lu, Q.R. et al. Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection. Cell 109, 75–86 (2002).
Zhou, Q., Choi, G. & Anderson, D.J. The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31, 791–807 (2001).
Shirasaki, R. & Pfaff, S.L. Transcriptional codes and the control of neuronal identity. Annu. Rev. Neurosci. 25, 251–281 (2002).
Arber, S. et al. Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 23, 659–674 (1999).
Lumsden, A. & Krumlauf, R. Patterning the vertebrate neuraxis. Science 274, 1109–1115 (1996).
Liu, J.P., Laufer, E. & Jessell, T.M. Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron 32, 997–1012 (2001).
Gao, B.X., Cheng, G. & Ziskind-Conhaim, L. Development of spontaneous synaptic transmission in the rat spinal cord. J. Neurophysiol. 79, 2277–2287 (1998).
Miles, G.B. et al. Functional properties of motoneurons derived from mouse embryonic stem cells. J. Neurosci. 24, 7848–7858 (2004).
Temple, S. The development of neural stem cells. Nature 414, 112–117 (2001).
Gabay, L., Lowell, S., Rubin, L.L. & Anderson, D.J. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40, 485–499 (2003).
Zhang, S.C. Embryonic stem cells for neural replacement therapy: prospects and challenges. J. Hematother. Stem Cell Res. 12, 625–634 (2003).
Ginis, I. et al. Differences between human and mouse embryonic stem cells. Dev. Biol. 269, 360–380 (2004).
Du, Z.W. & Zhang, S.C. Neural differentiation from embryonic stem cells: which way? Stem Cells Dev. 13, 372–381 (2004).
Kudoh, T., Wilson, S.W. & Dawid, I.B. Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129, 4335–4346 (2002).
Acknowledgements
This study was supported by the Amyotrophic Lateral Sclerosis Association, Hope for ALS, National Institutes of Health (National Institute of Neurological Disorders and Stroke, R01-NS045926), and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352). We thank M. Nakafuku, S. Pfaff and F. Vaccarino for generously providing antibodies against Olig2, Islet1/2 and Otx2, E. Terasawa for providing the embryonic monkey tissues and C.N. Svendsen and A. Bhattacharyya for reading the manuscript.
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Supplementary information
Supplementary Fig. 1
Expression of Pax6 and Sox1 mRNA by neuroectodermal cells. (PDF 44 kb)
Supplementary Fig. 2
Expression of Pax7 by neuroectodermal cells. (PDF 52 kb)
Supplementary Fig. 3
Expression of ChAT protein by motoneurons. (PDF 53 kb)
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Li, XJ., Du, ZW., Zarnowska, E. et al. Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23, 215–221 (2005). https://doi.org/10.1038/nbt1063
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DOI: https://doi.org/10.1038/nbt1063
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