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Primary culture of chick, mouse or human neural crest cells

Nature Protocols volume 6, pages 1568–1577 (2011)Cite this article

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Abstract

A highly enriched population of neural crest cells (NCCs) from amniote embryos, such as from chicks, mice and humans, is desirable for experiments in fate determination. NCCs are also useful for testing the functional effects of molecular changes underlying numerous human diseases of neural crest derivatives and for investigating their potential for therapeutic compensation. This protocol details embryonic microdissection followed by neural tube explantation. Conditions favoring NCC expansion and the maintenance of their stem cell–like properties are described. Although neural crest–like cells can be derived from a number of sites in the mature organism, full potential is best ensured by their purification from their source tissue at the outset of migration. Going from embryo to established cell line takes 4 d; the first is the most labor-intensive day, but minimal intervention is required thereafter.

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Figure 1: Some of the equipment required for this protocol.
Figure 2: Dissection of neural tube fragments from mouse and chick for explantation.
Figure 3: Examples of explanted neural tubes.

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Change history

  • 11 October 2013

     In the version of this article initially published, the descriptions of how to make FGF and complete NCC culture medium were incorrect. The text initially read "FGF (2.5 µg ml–1): Use 4 ml 10% (wt/vol) BSA solution to dissolve 25 µg of FGF." In addition, in the details for making complete NCC culture medium, the text initially read "Add 1 µl of EGF (2.5 µg ml–1) and 8 µl of FGF (10 µg ml–1)." The phrase about FGF should have read "FGF (2.5 µg ml–1): Use 4 ml 10% (wt/vol) BSA solution to dissolve 10 µg of FGF." For complete NCC culture medium, the text should have read "Add 1 µl of EGF (10 µg ml–1) and 8 µl of FGF (2.5 µg ml–1)." The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Le Douarin, N. & Kalcheim, C. The Neural Crest (Cambridge University Press, 1999).

  2. Roux, W. Beiträge zur entzicklungsmechanik des embryo. Zeitschrift fuer Biologie 21, 411 (1885).

    Google Scholar 

  3. Harrison, R.G. The outgrowth of the nerve fiber as a mode of protoplasmic movement. J. Exper. Zool. 9, 787–846 (1910).

    Article  Google Scholar 

  4. Pannett, C.A. & Compton, A. The cultivation of tissues in saline embryonic juice. Lancet 203, 381–384 (1924).

    Article  Google Scholar 

  5. Sieber-Blum, M. & Cohen, A. Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells. Dev. Biol. 80, 96–106 (1980).

    Article  CAS  PubMed  Google Scholar 

  6. Smith-Thomas, L.C. & Fawcett, J.W. Expression of Schwann cell markers by mammalian neural crest cells in vitro. Development 105, 251–262 (1989).

    CAS  PubMed  Google Scholar 

  7. Baroffio, A., Dupin, E. & Le Douarin, N.M. Common precursors for neural and mesectodermal derivatives in the cephalic neural crest. Development 112, 301–305 (1991).

    CAS  PubMed  Google Scholar 

  8. Boot, M.J. et al. Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries. Anat. Rec. 275, 1009–1018 (2003).

    Article  Google Scholar 

  9. Morrison, S.J. et al. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 96, 737–749 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Perris, R. et al. Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen. J. Cell. Sci. 106, 1357–1368 (1993).

    CAS  PubMed  Google Scholar 

  11. Stemple, D.L. & Anderson, D.J. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 71, 973–985 (1992).

    Article  CAS  PubMed  Google Scholar 

  12. Karbanová, J. et al. Characterization of dental pulp stem cells from impacted third molars cultured in low serum-containing medium. Cells Tissues Organs 193, 1–22 (2010).

    Google Scholar 

  13. Miura, M. et al. SHED: stem cells from human exfoliated deciduous teeth. PNAS 100, 5807–5812 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Stevens, A. et al. Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities. Stem Cells Dev. 17, 1175–1184 (2008).

    Article  PubMed  Google Scholar 

  15. Coura, G.S. et al. Human periodontal ligament: a niche of neural crest stem cells. J. Periodontal Res. 43, 531–536 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Kruger, G.M. et al. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron 35, 657–669 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li, H.-Y., Say, E.H.M. & Zhou, X.-F. Isolation and characterization of neural crest progenitors from adult dorsal root ganglia. Stem Cells 25, 2053–2065 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Coulpier, F. et al. Novel features of boundary cap cells revealed by the analysis of newly identified molecular markers. Glia 57, 1450–1457 (2009).

    Article  PubMed  Google Scholar 

  19. Lee, G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotech. 25, 1468–1475 (2007).

    Article  CAS  Google Scholar 

  20. Kawaguchi, J. et al. Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos. Development 137, 693–704 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Nagoshi, N. et al. Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell 2, 392–403 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Sviderskaya, E.V. et al. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells. FASEB J. 23, 3179–3192 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Biernaskie, J.A. et al. Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat. Protoc. 1, 2803–2812 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Wong, C.E. et al. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J. Cell Biol. 175, 1005–1015 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sieber-Blum, M. et al. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258–269 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Fernandes, K.J.L. et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat. Cell Biol. 6, 1082–1093 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Clewes, O. et al. Human epidermal neural crest stem cells (hEPI-NCSC)-characterization and directed differentiation into osteocytes and melanocytes. Stem Cell Rev. published online, 10.1007/s12015-011-9255-5 (1 April 2011).

  28. Abzhanov, A. et al. Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro. Development 130, 4567–4579 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. McGonnell, I.M. & Graham, A. Trunk neural crest has skeletogenic potential. Curr. Biol. 12, 767–771 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Fontaine-Pérus, J. & Chéraud, Y. Mouse-chick neural chimeras. Int. J. Dev. Biol. 49, 349–353 (2005).

    Article  PubMed  Google Scholar 

  31. de Pontual, L. et al. Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease. PNAS 106, 13921–13926 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Thomas, S. et al. Human neural crest cells display molecular and phenotypic hallmarks of stem cells. Hum. Mol. Genet. 17, 3411–3425 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lahav, R. et al. Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro. PNAS 95, 14214–14219 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dahéron, L. et al. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 22, 770–778 (2004).

    Article  PubMed  Google Scholar 

  35. Wakamatsu, Y. et al. Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation. Development 124, 1953–1962 (1997).

    CAS  PubMed  Google Scholar 

  36. Hu, Y.F., Zhang, Z.-J. & Sieber-Blum, M. An epidermal neural crest stem cell (EPI-NCSC) molecular signature. Stem Cells 24, 2692–2702 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Rao, M.S. & Anderson, D.J. Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells. J. Neurobiol. 32, 722–746 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Terayama, K. et al. Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1. PNAS 94, 6093–6098 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bakker, H. et al. Expression cloning of a cDNA encoding a sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope. J. Biol. Chem. 272, 29942–29946 (1997).

    Article  CAS  PubMed  Google Scholar 

  40. Ito, K., Morita, T. & Sieber-Blum, M. In vitro clonal analysis of mouse neural crest development. Dev. Biol. 157, 517–525 (1993).

    Article  CAS  PubMed  Google Scholar 

  41. Garcez, R.C. et al. Epidermal growth factor (EGF) promotes the in vitro differentiation of neural crest cells to neurons and melanocytes. Cell Mol. Neurobiol. 29, 1087–1091 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Pla, P. et al. Ednrb2 orients cell migration towards the dorsolateral neural crest pathway and promotes melanocyte differentiation. Pigment Cell Res. 18, 181–187 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Campos, P.B. et al. Chromosomal spread preparation of human embryonic stem cells for karyotyping. JoVE doi:10.3791/1512 (2009).

  44. Theiler, K. The House Mouse: Atlas of Embryonic Development (Springer-Verlag, 1989).

  45. Hamburger, V. & Hamilton, H.L. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272 (1992).

    Article  CAS  PubMed  Google Scholar 

  46. O'Rahilly, R. & Müller, F. Developmental Stages in Humans: Including a Revision of Streeter's 'Horizons' and a Survey of the Carnegie Collection (Carnegie Institution of Washington, 1987).

  47. White, R.M. et al. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471, 518–522 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Echelard, Y., Vassileva, G. & McMahon, A.P. Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development 120, 2213–2224 (1994).

    CAS  PubMed  Google Scholar 

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Acknowledgements

H.E. has been supported by the Sturge-Weber foundation (2001), the INSERM Avenir program (2002–2005), the Fondation pour la Recherche Médicale (DEQ20071210511), the Agence Nationale pour la Recherche (ANR2007-CRANIRARE) and Nevus Outreach. In addition to all the authors who have developed the neural crest culture protocols referenced herein and to the directors of INSERM U781 and U910 who have made it possible for me to obtain embryonic tissues over the years, I extend my particular gratitude to E. Dupin, C. Glavieux-Pardanaud, A. Gonçalves-Trentin, S. Thomas and C. Ziller for aiding me in optimizing conditions.

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  1. Institut National de la Santé et de la Recherche Médicale (INSERM) U910, Université de la Méditerranée Faculté de Médecine, Marseille, France

    Heather Etchevers

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Correspondence to Heather Etchevers.

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Etchevers, H. Primary culture of chick, mouse or human neural crest cells. Nat Protoc 6, 1568–1577 (2011). https://doi.org/10.1038/nprot.2011.398

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