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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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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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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