Fully Defined and Xeno-Free Induction of hPSCs into Neural Crest Using Top-Down Inhibition of BMP Signaling

  • James O. S. HacklandEmail author
  • Tom J. R. Frith
  • Peter W. Andrews
Part of the Methods in Molecular Biology book series (MIMB, volume 1976)


The neural crest is a transient embryonic tissue that originates from the border of the neural plate prior to delamination and migration throughout the developing embryo, where it contributes to a wide range of different tissues. Defects in neural crest development have been implicated in a variety of different disorders (neurocristopathies) including cancers, neuropathies, craniofacial malformations, and pigment disorders. The differentiation of human pluripotent stem cells (hPSCs) into an in vitro counterpart to neural crest cells holds huge potential for the study of neural crest development, as well as modeling neurocristopathy, carrying out drug discovery experiments and eventually cell replacement therapy. Here we describe a method for generating human neural crest cells from hPSCs that is fully defined and free from animal-derived components. We found that in the absence of serum or bovine serum albumin (BSA), variability in endogenous BMP expression leads to unpredictable differentiation efficiency. In order to control against this issue, we have developed a system termed “top-down inhibition” (TDi) that allows robust neural crest induction as described below.

Key words

Neural crest BMP Ectoderm Human pluripotent stem cells Embryonic stem cells Top-down inhibition TDi Endogenous signal control 



The H9: SOX10 BAC hESC line used in the development of this protocol and in generating the data presented in Fig. 2 was kindly provided by Lorenz Studer, Mark Tomishima, and Yvonne Mica.

Funding: This work was funded by grants from the Medical Research Council (R/143416-14-1) and the European Community’s Seventh Framework Programme (FP7/2007-2013 under grant agreement no 602423).


  1. 1.
    Vazin T, Chen J, Lee C-T, Amable R, Freed WJ (2005) Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells 23(7):923–930CrossRefGoogle Scholar
  2. 2.
    Lee G, Chambers SM, Tomishima MJ, Studer L (2010) Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc 5(4):688–701CrossRefGoogle Scholar
  3. 3.
    Mica Y, Lee G, Chambers SM, Tomishima MJ, Studer L (2013) Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Rep 3(4):1140–1152CrossRefGoogle Scholar
  4. 4.
    Menendez L, Yatskievych TA, Antin PB, Dalton S (2011) Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc Natl Acad Sci U S A 108(48):19240–19245CrossRefGoogle Scholar
  5. 5.
    Leung AW, Murdoch B, Salem AF, Prasad MS, Gomez GA, García-Castro MI (2016) WNT/beta-catenin signaling mediates human neural crest induction via a pre-neural border intermediate. Development 143(3):398–410CrossRefGoogle Scholar
  6. 6.
    Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Y, Helms J, Chang C-P, Zhao Y, Swigut T, Wysocka J (2010) CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463(7283):958–962CrossRefGoogle Scholar
  7. 7.
    Chambers SM, Qi Y, Mica Y, Lee G, Zhang X-J, Niu L, Bilsland J, Cao L, Stevens E, Whiting P, Shi S-H, Studer L (2012) Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol 30(7):715–720CrossRefGoogle Scholar
  8. 8.
    Hackland JOS, Frith TJR, Thompson O, Marin Navarro A, García-Castro MI, Unger C, Andrews PW (2017) Top-down inhibition of BMP signaling enables robust induction of hPSCs into neural crest in fully defined, Xeno-free conditions. Stem Cell Reports 9(4):1043–1052CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • James O. S. Hackland
    • 1
    • 2
    Email author
  • Tom J. R. Frith
    • 2
  • Peter W. Andrews
    • 2
  1. 1.Department of Biomedical SciencesUniversity of California, RiversideRiversideUSA
  2. 2.Department of Biomedical ScienceUniversity of SheffieldSheffieldUK

Personalised recommendations