Derivation of neural crest cells from human pluripotent stem cells

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Multipotentiality of NC cells in human body.
Figure 2: Distinct NC precursor fates along the anterior–posterior axis of the embryo.
Figure 3: Multistep differentiation of NC cells from hPSCs in vitro.
Figure 4
Figure 5: Procedures for isolating hESC-derived neural crest cells with MS5 coculture.
Figure 6: Target hESC/hiPSC density for NSB induction toward NC cells.
Figure 7: NC cell marker expression using NSB induction.
Figure 8: Representative image of FACS isolation for NC cells.
Figure 9: Propagation of sorted NC cells.
Figure 10: Specification of hESC-NC cells toward peripheral neurons.
Figure 11: Schwann cell differentiation from hESC-NC cells.
Figure 12: Representative morphology of myofibroblast cells derived from hESC-NC cells.
Figure 13: Adipogenic, chondrogenic and osteogenic differentiation of myofibroblast cells derived from hESC-NC cells.

References

  1. 1

    Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    Article  Google Scholar 

  4. 4

    Roy, N.S. et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259–1268 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    CAS  Article  PubMed  Google Scholar 

  6. 6

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

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Pruszak, J., Sonntag, K.C., Aung, M.H., Sanchez-Pernaute, R. & Isacson, O. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 25, 2257–2268 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes. Dev. 22, 152–165 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9

    Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    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 

  11. 11

    Slaugenhaupt, S.A. et al. Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. Am. J. Hum. Genet. 68, 598–605 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Farlie, P.G., McKeown, S.J. & Newgreen, D.F. The neural crest: basic biology and clinical relationships in the craniofacial and enteric nervous systems. Birth Defects Res. C Embryo Today 72, 173–189 (2004).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Wurdak, H., Ittner, L.M. & Sommer, L. DiGeorge syndrome and pharyngeal apparatus development. Bioessays 28, 1078–1086 (2006).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Etchevers, H.C., Amiel, J. & Lyonnet, S. Molecular bases of human neurocristopathies. Adv. Exp. Med. Biol. 589, 213–234 (2006).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Ross, R.A. & Spengler, B.A. Human neuroblastoma stem cells. Semin. Cancer. Biol. 17, 241–247 (2007).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Carniti, C. et al. The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease. Am. J. Pathol. 168, 1262–1275 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Hims, M.M. et al. A humanized IKBKAP transgenic mouse models a tissue-specific human splicing defect. Genomics 90, 389–396 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Selleck, M.A. & Bronner-Fraser, M. The genesis of avian neural crest cells: a classic embryonic induction. Proc. Natl. Acad. Sci. USA 93, 9352–9357 (1996).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Crane, J.F. & Trainor, P.A. Neural crest stem and progenitor cells. Annu. Rev. Cell. Dev. Biol. 22, 267–286 (2006).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Dupin, E., Real, C. & Ledouarin, N. The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3. An. Acad. Bras. Cienc. 73, 533–545 (2001).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Anderson, D.J. Genes, lineages and the neural crest: a speculative review. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355, 953–964 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Creuzet, S., Couly, G. & Le Douarin, N.M. Patterning the neural crest derivatives during development of the vertebrate head: insights from avian studies. J. Anat. 207, 447–459 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    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).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Buchstaller, J. et al. Efficient isolation and gene expression profiling of small numbers of neural crest stem cells and developing Schwann cells. J. Neurosci. 24, 2357–2365 (2004).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    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).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Sauka-Spengler, T., Meulemans, D., Jones, M. & Bronner-Fraser, M. Ancient evolutionary origin of the neural crest gene regulatory network. Dev. Cell 13, 405–420 (2007).

    CAS  Article  PubMed  Google Scholar 

  29. 29

    Holland, L.Z. & Holland, N.D. Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate? J. Anat. 199, 85–98 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Jiang, X. et al. Isolation and characterization of neural crest stem cells derived from in vitro-differentiated human embryonic stem cells. Stem. Cells. Dev. 18, 1059–1070 (2008).

    Article  PubMed Central  Google Scholar 

  31. 31

    Zhou, Y. & Snead, M.L. Derivation of cranial neural crest-like cells from human embryonic stem cells. Biochem. Biophys. Res. Commun. 376, 542–547 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Papapetrou, E.P. et al. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106, 12759–12764 (2009).

    CAS  Article  PubMed  Google Scholar 

  33. 33

    Lee, G. et al. Modeling pathogenesis and treatment of familial dysautonomia using patient specific iPS cells. Nature 461, 402–406 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34

    Nishikawa, S., Goldstein, R.A. & Nierras, C.R. The promise of human induced pluripotent stem cells for research and therapy. Nat. Rev. Mol. Cell. Biol. 9, 725–729 (2008).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Billon, N. et al. The generation of adipocytes by the neural crest. Development 134, 2283–2292 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36

    Sandell, L.L. & Trainor, P.A. Neural crest cell plasticity. Size matters. Adv. Exp. Med. Biol 589, 78–95 (2006).

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Barberi, T., Willis, L.M., 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 

  38. 38

    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 

  39. 39

    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 

  40. 40

    Placantonakis, D.G. et al. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells 27, 521–532 (2009).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Anderson, D.J. Cellular and molecular biology of neural crest cell lineage determination. Trends Genet. 13, 276–280 (1997).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

All authors contributed to the development of the method and the writing of the manuscript.

Corresponding author

Correspondence to Lorenz Studer.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

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