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Derivation of neural crest cells from human pluripotent stem cells

Nature Protocols volume 5, pages 688–701 (2010)Cite this article

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Abstract

Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-β signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for generating purified hPSC-NC precursors using this protocol is 2–5 weeks.

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Figure 1: Multipotentiality of NC cells in human body.
Figure 2: Distinct NC precursor fates along the anterior–posterior axis of the embryo.
Figure 3: Multistep differentiation of NC cells from hPSCs in vitro.
Figure 4
Figure 5: Procedures for isolating hESC-derived neural crest cells with MS5 coculture.
Figure 6: Target hESC/hiPSC density for NSB induction toward NC cells.
Figure 7: NC cell marker expression using NSB induction.
Figure 8: Representative image of FACS isolation for NC cells.
Figure 9: Propagation of sorted NC cells.
Figure 10: Specification of hESC-NC cells toward peripheral neurons.
Figure 11: Schwann cell differentiation from hESC-NC cells.
Figure 12: Representative morphology of myofibroblast cells derived from hESC-NC cells.
Figure 13: Adipogenic, chondrogenic and osteogenic differentiation of myofibroblast cells derived from hESC-NC cells.

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Acknowledgements

We thank all the members of Studer, Tabar and Tomishima labs for technical advice and helpful discussions on the protocol. Our work was supported by grants from the Starr Foundation, NYSTEM and NINDS. Additional support was provided by the New York Stem Cell Foundation (NYCSF, Druckenmiller fellowships to G.L.) and by the Starr Scholar fellowship to S.M.C.

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Authors and Affiliations

  1. Developmental Biology Program, Sloan-Kettering Institute, New York, NY, USA

    Gabsang Lee, Stuart M Chambers, Mark J Tomishima & Lorenz Studer

  2. Center for Stem Cell Biology, Sloan-Kettering Institute, New York, NY, USA

    Gabsang Lee, Stuart M Chambers, Mark J Tomishima & Lorenz Studer

  3. SKI Stem Cell Research Facility, Sloan-Kettering Institute, New York, NY, USA

    Mark J Tomishima & Lorenz Studer

  4. Department of Neurosurgery, Sloan-Kettering Institute, New York, NY, USA

    Lorenz Studer

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  1. Gabsang Lee
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All authors contributed to the development of the method and the writing of the manuscript.

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Correspondence to Lorenz Studer.

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Lee, G., Chambers, S., Tomishima, M. et al. Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc 5, 688–701 (2010). https://doi.org/10.1038/nprot.2010.35

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