Abstract
Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ∼15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.
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Acknowledgements
This work was supported by a grant to S.D. from the National Institute for General Medical Sciences (GM085354) and a grant to J.D.L. from the Children's Glaucoma Foundation.
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L.M. performed all differentiation experiments, analyzed the data and contributed to writing the manuscript; M.J.K. generated hiPSCs from fibroblasts and performed quality control analysis; S.S.P. maintained patient fibroblasts; A.T.P. designed and performed zebrafish in vivo experiments; J.D.L. designed zebrafish in vivo experiments; M.L.C. supervised the isolation of patient fibroblasts and obtained patient consent; S.D. provided overall direction for the project, analysis of data and writing of the manuscript.
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Menendez, L., Kulik, M., Page, A. et al. Directed differentiation of human pluripotent cells to neural crest stem cells. Nat Protoc 8, 203–212 (2013). https://doi.org/10.1038/nprot.2012.156
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DOI: https://doi.org/10.1038/nprot.2012.156
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