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Differentiation of spinal motor neurons from pluripotent human stem cells

Nature Protocols volume 4, pages 1295–1304 (2009)Cite this article

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.

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Figure 1: Scheme of differentiation of spinal cord motoneurons (MNs) from hESCs.
Figure 2: Induction of neuroepithelial cells.
Figure 3: Patterning of spinal cord progenitors.
Figure 4: Differentiation and maturation of hESC-derived motoneurons.

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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).

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Authors and 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

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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.

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Correspondence to Su-Chun Zhang.

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Hu, BY., Zhang, SC. Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4, 1295–1304 (2009). https://doi.org/10.1038/nprot.2009.127

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