Neural Crest Cell Models of Development and Toxicity: Cytotoxicity Assay Using Human Pluripotent Stem Cell-Derived Cranial Neural Crest Cell Model

  • Mika Suga
  • Miho K. Furue
Part of the Methods in Molecular Biology book series (MIMB, volume 1965)


Cranial neural crest cells (NCCs) migrate to the branchial arches and give rise to the majority of cranial mesenchyme that eventually differentiates into odontoblasts, cartilage, craniofacial bone, and connective tissue; a subset of these cells differentiate into cranial ganglia. Here we present a protocol that describes directed differentiation method of human pluripotent stem cells into cranial NCC-like cells and a cytotoxicity assay using hPSC-derived cranial NCC-like cells. This cell-based assay system allows for high-sensitive cytotoxicity detection of test chemicals. These methods can be applied to predict drug/chemical toxicity effect on early craniofacial development.

Key words

Cranial neural crest cells Cranial mesenchyme Human pluripotent stem cells Human-induced pluripotent stem cells Defined culture medium Cytotoxicity assay 



This work was supported by JSPS KAKENHI Grant Numbers 16H05535 and 00340448 to M.S. and M.K.F. and Research Project for Practical Applications of Regenerative Medicine from the Ministry of Health, Labor and Welfare of Japan and the Japan Agency for Medical Research and Development (AMED) to M.K.F. Authors thank Yu Jung Liu, Takako Nakano, and Naoko Ueda for their excellent technical supports.


  1. 1.
    Mossey P (2007) Epidemiology underpinning research in the aetiology of orofacial clefts. Orthod Craniofac Res 10(3):114–120. Scholar
  2. 2.
    Gross JB, Hanken J (2008) Review of fate-mapping studies of osteogenic cranial neural crest in vertebrates. Dev Biol 317(2):389–400. Scholar
  3. 3.
    Santagati F, Rijli FM (2003) Cranial neural crest and the building of the vertebrate head. Nat Rev Neurosci 4(10):806–818. Scholar
  4. 4.
    Sipes NS, Martin MT, Reif DM, Kleinstreuer NC, Judson RS, Singh AV, Chandler KJ, Dix DJ, Kavlock RJ, Knudsen TB (2011) Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data. Toxicol Sci 124(1):109–127. Scholar
  5. 5.
    Seiler AE, Spielmann H (2011) The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6(7):961–978. Scholar
  6. 6.
    Jensen J, Hyllner J, Bjorquist P (2009) Human embryonic stem cell technologies and drug discovery. J Cell Physiol 219(3):513–519. Scholar
  7. 7.
    Krtolica A, Giritharan G (2010) Use of human embryonic stem cell-based models for male reproductive toxicity screening. Syst Biol Reprod Med 56(3):213–221. Scholar
  8. 8.
    Krug AK, Kolde R, Gaspar JA, Rempel E, Balmer NV, Meganathan K, Vojnits K, Baquie M, Waldmann T, Ensenat-Waser R, Jagtap S, Evans RM, Julien S, Peterson H, Zagoura D, Kadereit S, Gerhard D, Sotiriadou I, Heke M, Natarajan K, Henry M, Winkler J, Marchan R, Stoppini L, Bosgra S, Westerhout J, Verwei M, Vilo J, Kortenkamp A, Hescheler J, Hothorn L, Bremer S, van Thriel C, Krause KH, Hengstler JG, Rahnenfuhrer J, Leist M, Sachinidis A (2013) Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Arch Toxicol 87(1):123–143. Scholar
  9. 9.
    Nakamura Y, Matsuo J, Miyamoto N, Ojima A, Ando K, Kanda Y, Sawada K, Sugiyama A, Sekino Y (2014) Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study. J Pharmacol Sci 124(4):494–501CrossRefGoogle Scholar
  10. 10.
    Kanda Y, Yamazaki D, Kurokawa J, Inutsuka T, Sekino Y (2016) Points to consider for a validation study of iPS cell-derived cardiomyocytes using a multi-electrode array system. J Pharmacol Toxicol Methods 81:196–200. Scholar
  11. 11.
    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 108(48):19240–19245. Scholar
  12. 12.
    Menendez L, Kulik MJ, Page AT, Park SS, Lauderdale JD, Cunningham ML, Dalton S (2013) Directed differentiation of human pluripotent cells to neural crest stem cells. Nat Protoc 8(1):203–212. Scholar
  13. 13.
    Fukuta M, Nakai Y, Kirino K, Nakagawa M, Sekiguchi K, Nagata S, Matsumoto Y, Yamamoto T, Umeda K, Heike T, Okumura N, Koizumi N, Sato T, Nakahata T, Saito M, Otsuka T, Kinoshita S, Ueno M, Ikeya M, Toguchida J (2014) Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media. PLoS One 9(12):e112291. Scholar
  14. 14.
    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):1140–1152. Scholar
  15. 15.
    Hackland JOS, Frith TJR, Thompson O, Marin Navarro A, Garcia-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):1043–1052. Scholar
  16. 16.
    Mimura S, Suga M, Okada K, Kinehara M, Nikawa H, Furue MK (2016) Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells. Int J Dev Biol 60(1–3):21–28. Scholar
  17. 17.
    Suga M, Hayashi Y, Furue MK (2017) In vitro models of cranial neural crest development toward toxicity tests: frog, mouse, and human. Oral Dis 23(5):559–565. Scholar
  18. 18.
    Kumagai A, Suga M, Yanagihara K, Itoh Y, Takemori H, Furue MK (2016) A simple method for labeling human embryonic stem cells destined to lose undifferentiated potency. Stem Cells Transl Med 5(3):275–281. Scholar
  19. 19.
    Mimura S, Kimura N, Hirata M, Tateyama D, Hayashida M, Umezawa A, Kohara A, Nikawa H, Okamoto T, Furue MK (2011) Growth factor-defined culture medium for human mesenchymal stem cells. Int J Dev Biol 55(2):181–187. Scholar
  20. 20.
    Mine Y, Makihira S, Yamaguchi Y, Tanaka H, Nikawa H (2014) Involvement of ERK and p38 MAPK pathways on Interleukin-33-induced RANKL expression in osteoblastic cells. Cell Biol Int 38(5):655–662. Scholar
  21. 21.
    Mine Y, Makihira S, Nikawa H, Murata H, Hosokawa R, Hiyama A, Mimura S (2010) Impact of titanium ions on osteoblast-, osteoclast- and gingival epithelial-like cells. J Prosthodont Res 54(1):1–6. Scholar
  22. 22.
    Initiative ISCB (2009) Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes. Stem Cell Rev 5(4):301–314. Scholar
  23. 23.
    Furue MK, Na J, Jackson JP, Okamoto T, Jones M, Baker D, Hata R, Moore HD, Sato JD, Andrews PW (2008) Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci U S A 105(36):13409–13414. Scholar
  24. 24.
    Wang L, Schulz TC, Sherrer ES, Dauphin DS, Shin S, Nelson AM, Ware CB, Zhan M, Song CZ, Chen X, Brimble SN, McLean A, Galeano MJ, Uhl EW, D’Amour KA, Chesnut JD, Rao MS, Blau CA, Robins AJ (2007) Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110(12):4111–4119. Scholar

Copyright information

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

Authors and Affiliations

  • Mika Suga
    • 1
  • Miho K. Furue
    • 1
  1. 1.Laboratory of Stem Cell CulturesNational Institutes of Biomedical Innovation, Health and NutritionOsakaJapan

Personalised recommendations