Protocol | Published:

Differentiation of spinal motor neurons from pluripotent human stem cells

Nature Protocols volume 4, pages 12951304 (2009) | Download Citation

Subjects

Abstract

We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic, HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8–10 weeks for the production of functional mature motoneurons. In comparison with other methods, our protocol does not use feeder cells, has a minimum dependence on proteins (purmorphamine replacing SHH), has controllable adherent selection and is adaptable for scalable suspension culture.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , & In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

  2. 2.

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

  3. 3.

    et al. Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp. Neurol. 196, 224–234 (2005).

  4. 4.

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

  5. 5.

    et al. Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23, 781–790 (2005).

  6. 6.

    et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259–1268 (2006).

  7. 7.

    et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288–296 (2005).

  8. 8.

    , , & Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture. J. Neurosci. 27, 3069–3077 (2007).

  9. 9.

    , , , & Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452 (2009).

  10. 10.

    , , , & Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49, 385–396 (2005).

  11. 11.

    Neural subtype specification from embryonic stem cells. Brain Pathol. 16, 132–142 (2006).

  12. 12.

    et al. Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system. Neuron 25, 317–329 (2000).

  13. 13.

    , & Identification of a novel family of oligodendrocyte lineage-specific basic helix–loop–helix transcription factors. Neuron 25, 331–343 (2000).

  14. 14.

    et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90, 169–180 (1997).

  15. 15.

    et al. Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31, 757–771 (2001).

  16. 16.

    , , & Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

  17. 17.

    , , & Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. Genes Dev. 19, 282–294 (2005).

  18. 18.

    & Olig transcription factors are expressed in oligodendrocyte and neuronal cells in human fetal CNS. J. Neurosci. 25, 10064–10073 (2005).

  19. 19.

    et al. Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin. J. Comp. Neurol. 266, 332–359 (1987).

  20. 20.

    et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells 25, 1511–1520 (2007).

  21. 21.

    et al. Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells 26, 886–893 (2008).

  22. 22.

    Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells 27, 806–811 (2009).

  23. 23.

    et al. Regulation of neural specification from human embryonic stem cells by BMP and FGF. Stem Cells doi: 10.1002/stem.99 (2009).

  24. 24.

    , , & Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev. Biol. 275, 124–142 (2004).

  25. 25.

    et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 100, 5828–5833 (2003).

  26. 26.

    & Differentiation of neuroepithelia from human embryonic stem cells. Methods in Molecular Biology 549 (eds. Scolding, N.J. & Gordon, D.) 51–58 (Humana Press, New York, 2009).

Download references

Acknowledgements

This study was supported by the National Institutes of Neurological Diseases and Stroke (NS045926 and NS057778), the ALS Association and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352).

Author information

Affiliations

  1. Department of Anatomy and Department of Neurology, School of Medicine and Public Health, Waisman Center, The WiCell Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA.

    • Bao-Yang Hu
    •  & Su-Chun Zhang

Authors

  1. Search for Bao-Yang Hu in:

  2. Search for Su-Chun Zhang in:

Contributions

B.-Y.H. designed and performed experiments, analyzed data and wrote the paper; S.-C.Z. supervised the project, designed experiments, analyzed data, wrote and approved the final paper.

Corresponding author

Correspondence to Su-Chun Zhang.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2009.127

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.