Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Nature Biotechnology volume 25, pages 1468–1475 (2007)Cite this article

A Corrigendum to this article was published on 01 July 2008

This article has been updated

Abstract

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest–related disorders.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cells expressing neural crest markers are present in cultures of hES cell–derived neural rosettes and can be induced through extrinsic cues.
Figure 2: Molecular characterization of neural crest precursor cells in hES cell–derived neural rosettes.
Figure 3: Identification of neural crest stem cell potential by clonal assay and sphere-forming assay.
Figure 4: Differentiation of hES cell–derived NCS cells toward peripheral nervous system lineages.
Figure 5: Characterization and differentiation of mesenchymal precursor cells derived from hES cell–derived NCS cells.
Figure 6: In vivo transplantation of hES cell–derived NCS cell progeny.
Figure 7: Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell.

Change history

  • 08 July 2008

    In the version of this article initially published, a reference was missing from the first paragraph. The reference (no. 6) has been added and subsequent references renumbered in the HTML and PDF versions of the article.

References

  1. Joseph, N.M. & Morrison, S.J. Toward an understanding of the physiological function of Mammalian stem cells. Dev. Cell 9, 173–183 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Perrier, A.L. et al. From the Cover: Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li, X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005).

    Article  PubMed  Google Scholar 

  5. Lee, H.J. et al. Directed differentiation and transplantation of human embryonic stem cell derived motoneurons. Stem Cells 25, 1931–1939 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Lazzari, G. et al. Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro. Stem Cells 24, 2514–2521 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B. & Goldstein, R.S. Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells. Stem Cells 23, 923–930 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Fang, D. et al. Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells 24, 1668–1677 (2006).

    Article  PubMed  Google Scholar 

  9. Morrison, S.J., White, P.M., Zock, C. & Anderson, D.J. 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. 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 

  11. Yoshida, S. et al. Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea. Stem Cells 24, 2714–2722 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Gitler, A.D., Brown, C.B., Kochilas, L., Li, J. & Epstein, J.A. Neural crest migration and mouse models of congenital heart disease. Cold Spring Harb. Symp. Quant. Biol. 67, 57–62 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Iwashita, T., Kruger, G.M., Pardal, R., Kiel, M.J. & Morrison, S.J. Hirschsprung disease is linked to defects in neural crest stem cell function. Science 301, 972–976 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fuchs, S. & Sommer, L. The neural crest: understanding stem cell function in development and disease. Neurodegener. Dis. 4, 6–12 (2007).

    Article  PubMed  Google Scholar 

  15. Edery, P. et al. Mutations of the RET proto-oncogene in Hirschsprung's disease. Nature 367, 378–380 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Pingault, V. et al. SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet. 18, 171–173 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Bixby, S., Kruger, G.M., Mosher, J.T., Joseph, N.M. & Morrison, S.J. Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35, 643–656 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Fedtsova, N.G. & Turner, E.E. Brn-3.0 expression identifies early post-mitotic CNS neurons and sensory neural precursors. Mech. Dev. 53, 291–304 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Molofsky, A.V. et al. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature 425, 962–967 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Molne, M. et al. Early cortical precursors do not undergo LIF-mediated astrocytic differentiation. J. Neurosci. Res. 59, 301–311 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Song, M.R. & Ghosh, A. FGF2-induced chromatin remodeling regulates CNTF-mediated gene expression and astrocyte differentiation. Nat. Neurosci. 7, 229–235 (2004).

    Article  PubMed  Google Scholar 

  22. Shimozaki, K., Namihira, M., Nakashima, K. & Taga, T. Stage- and site-specific DNA demethylation during neural cell development from embryonic stem cells. J. Neurochem. 93, 432–439 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Fan, G. et al. DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling. Development 132, 3345–3356 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Noden, D.M. & Trainor, P.A. Relations and interactions between cranial mesoderm and neural crest populations. J. Anat. 207, 575–601 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kang, P. & Svoboda, K.K. Epithelial-mesenchymal transformation during craniofacial development. J. Dent. Res. 84, 678–690 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Baroffio, A., Dupin, E. & Le Douarin, N.M. Clone-forming ability and differentiation potential of migratory neural crest cells. Proc. Natl. Acad. Sci. USA 85, 5325–5329 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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 

  28. Barberi, T., Willis, L., Socci, N.D. & Studer, L. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med. 2, e161 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Barberi, T. et al. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat. Med. 13, 642–648 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M. & Yoo, J.U. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Knecht, A.K. & Bronner-Fraser, M. Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3, 453–461 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Le Douarin, N.M. & Dupin, E. Multipotentiality of the neural crest. Curr. Opin. Genet. Dev. 13, 529–536 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Sailer, M.H. et al. BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells. J. Cell Sci. 118, 5849–5860 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Mizuseki, K. et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 100, 5828–5833 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Marzi, I. et al. Purging of the neuroblastoma stem cell compartment and tumor regression on exposure to hypoxia or cytotoxic treatment. Cancer Res. 67, 2402–2407 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Motohashi, T., Aoki, H., Chiba, K., Yoshimura, N. & Kunisada, T. Multipotent cell fate of neural crest-like cells derived from embryonic stem cells. Stem Cells 25, 402–410 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Yamane, T., Hayashi, S., Mizoguchi, M., Yamazaki, H. & Kunisada, T. Derivation of melanocytes from embryonic stem cells in culture. Dev. Dyn. 216, 450–458 (1999).

    Article  CAS  PubMed  Google Scholar 

  39. Wilson, Y.M., Richards, K.L., Ford-Perriss, M.L., Panthier, J.J. & Murphy, M. Neural crest cell lineage segregation in the mouse neural tube. Development 131, 6153–6162 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. White, P.M. et al. Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron 29, 57–71 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Kalcheim, C. & Burstyn-Cohen, T. Early stages of neural crest ontogeny: formation and regulation of cell delamination. Int. J. Dev. Biol. 49, 105–116 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Kondo, T. & Raff, M. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289, 1754–1757 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Bhattacherjee, V. et al. Neural crest and mesoderm lineage-dependent gene expression in orofacial development. Differentiation 75, 463–477 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Etchevers, H.C., Vincent, C., Le Douarin, N.M. & Couly, G.F. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 128, 1059–1068 (2001).

    CAS  PubMed  Google Scholar 

  45. Korn, J., Christ, B. & Kurz, H. Neuroectodermal origin of brain pericytes and vascular smooth muscle cells. J. Comp. Neurol. 442, 78–88 (2002).

    Article  PubMed  Google Scholar 

  46. Trainor, P.A., Ariza-McNaughton, L. & Krumlauf, R. Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning. Science 295, 1288–1291 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Abzhanov, A., Tzahor, E., Lassar, A.B. & Tabin, C.J. 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 

  48. James, D., Noggle, S.A., Swigut, T. & Brivanlou, A.H. Contribution of human embryonic stem cells to mouse blastocysts. Dev. Biol. 295, 90–102 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Goldstein, R.S. Transplantation of human embryonic stem cells to the chick embryo. Methods Mol. Biol. 331, 137–151 (2006).

    PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Z. Dincer and M. Tomishima for technical advice and the Tri-institutional Stem Cell Research Facility at the Memorial Sloan-Kettering Cancer Center (MSKCC) for help in the time-lapse studies. We also would like to thank J. Itskovitz and M. Amit for providing the I-8 cell line, the MSKCC genomics core for performing microarray hybridizations, and members of the Studer, Tabar and Tomishima labs for helpful discussions. This work was supported through the Tri-Institutional Stem Cell Initiative funded by the Starr Foundation.

Author information

Author notes

  1. Hyesoo Kim and Yechiel Elkabetz: These authors contributed equally to this work.

Authors and Affiliations

  1. Developmental Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, 10021, New York, USA

    Gabsang Lee, Hyesoo Kim, Yechiel Elkabetz & Lorenz Studer

  2. Department of Neurosurgery, Sloan-Kettering Institute, 1275 York Ave., New York, 10021, New York, USA

    George Al Shamy, Georgia Panagiotakos, Viviane Tabar & Lorenz Studer

  3. Division of Neuroscience, Beckman Research Institute of The City of Hope, 1500 E. Duarte Road, Duarte, 91010, California, USA

    Tiziano Barberi

Authors
  1. Gabsang Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hyesoo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yechiel Elkabetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George Al Shamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Georgia Panagiotakos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tiziano Barberi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Viviane Tabar
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lorenz Studer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.L., T.B., V.T. and L.S. designed the study. G.L., V.T. and L.S. analyzed the data and wrote the manuscript. G.L., H.K., Y.E., G.A.S., G.P. and L.S. performed the experiments.

Corresponding author

Correspondence to Lorenz Studer.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6; Supplementary Methods (PDF 362 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, G., Kim, H., Elkabetz, Y. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol 25, 1468–1475 (2007). https://doi.org/10.1038/nbt1365

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1365

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing