Skip to main content

Directing Human Embryonic Stem Cells to a Retinal Fate

Part of the Methods in Molecular Biology book series (MIMB,volume 636)

Abstract

Substantial progress has been made in the development of methods to direct embryonic stem cells to differentiate into various regions of the central nervous system (CNS). We have used the current model of eye specification to develop a protocol for directing human embryonic stem cells to generate retinal progenitors and various types of retinal neurons. Our method uses a multistep protocol in which embryoid bodies are treated with inhibitors of both BMP signaling and canonical Wnt signaling to promote expression of key eye field transcription factors (EFTFs), as assayed by RT-PCR and immunofluorescence microscopy. The retinal progenitor cells spontaneously undergo differentiation into various types of retinal neurons, including photoreceptors, and this can be promoted by treatment with small molecule inhibitors of the Notch pathway.

Key words

  • Eye field
  • Retinal progenitor
  • Notch
  • Photoreceptor
  • hES cells
  • DAPT

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-60761-691-7_9
  • Chapter length: 15 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-60761-691-7
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   139.99
Price excludes VAT (USA)
Hardcover Book
USD   199.99
Price excludes VAT (USA)
Fig. 1.
Fig. 2.
Fig. 3.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Lamba, D., Karl, M., and Reh, T. (2008). Neural regeneration and cell replacement: a view from the eye. Cell Stem Cell 2, 538–549.

    CAS  CrossRef  PubMed  Google Scholar 

  2. Sanchez-Pernaute, R., Studer, L., Bankiewicz, K.S., Major, E.O., and McKay, R.D. (2001). In vitro generation and transplantation of precursor-derived human dopamine neurons. J Neurosci Res 65, 284–288.

    CAS  CrossRef  PubMed  Google Scholar 

  3. Wichterle, H., Lieberam, I., Porter, J.A., and Jessell, T.M. (2002). Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397.

    CAS  CrossRef  PubMed  Google Scholar 

  4. Cho, M.S., Hwang, D.Y., and Kim, D.W. (2008). Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nat Protoc 3, 1888–1894.

    CAS  CrossRef  PubMed  Google Scholar 

  5. Dimos, J.T., Rodolfa, K.T., Niakan, K.K., Weisenthal, L.M., Mitsumoto, H., Chung, W., Croft, G.F., Saphier, G., Leibel, R., Goland, R., et al. (2008). Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221.

    CAS  CrossRef  PubMed  Google Scholar 

  6. Park, C.H., Minn, Y.K., Lee, J.Y., Choi, D.H., Chang, M.Y., Shim, J.W., Ko, J.Y., Koh, H.C., Kang, M.J., Kang, J.S., et al. (2005). In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J Neurochem 92, 1265–1276.

    CAS  CrossRef  PubMed  Google Scholar 

  7. Niehrs, C. (2001). Developmental biology. Solving a sticky problem. Nature 413, 787–788.

    CAS  CrossRef  PubMed  Google Scholar 

  8. del Barco Barrantes, I., Davidson, G., Grone, H.J., Westphal, H., and Niehrs, C. (2003). Dkk1 and noggin cooperate in mammalian head induction. Genes Dev 17, 2239–2244.

    CrossRef  PubMed  Google Scholar 

  9. Zuber, M.E., Gestri, G., Viczian, A.S., Barsacchi, G., and Harris, W.A. (2003). Specification of the vertebrate eye by a network of eye field transcription factors. Development 130, 5155–5167.

    CAS  CrossRef  PubMed  Google Scholar 

  10. Cavodeassi, F., Carreira-Barbosa, F., Young, R.M., Concha, M.L., Allende, M.L., Houart, C., Tada, M., and Wilson, S.W. (2005). Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway. Neuron 47, 43–56.

    CAS  CrossRef  PubMed  Google Scholar 

  11. Esteve, P., and Bovolenta, P. (2006). Secreted inducers in vertebrate eye development: more functions for old morphogens. Curr Opin Neurobiol 16, 13–19.

    CAS  CrossRef  PubMed  Google Scholar 

  12. Lamba, D.A., Gust, J., and Reh, T.A. (2009). Transplantation of Human Embryonic Stem Cell-Derived Photoreceptors Restores Some Visual Function in Crx-Deficient Mice. Cell Stem Cell 4, 1–7.

    CrossRef  Google Scholar 

  13. Lamba, D.A., Karl, M.O., Ware, C.B., and Reh, T.A. (2006). Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A 103, 12769–12774.

    CAS  CrossRef  PubMed  Google Scholar 

  14. Nelson, B.R., Hartman, B.H., Georgi, S.A., Lan, M.S., and Reh, T.A. (2007). Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells. Dev Biol 304, 479–498.

    CAS  CrossRef  PubMed  Google Scholar 

  15. Nelson, B.R., and Reh, T.A. (2008). Relationship between Delta-like and proneural bHLH genes during chick retinal development. Dev Dyn 237, 1565–1580.

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Thomas A. Reh .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Reh, T.A., Lamba, D., Gust, J. (2010). Directing Human Embryonic Stem Cells to a Retinal Fate. In: Ding, S. (eds) Cellular Programming and Reprogramming. Methods in Molecular Biology, vol 636. Humana Press. https://doi.org/10.1007/978-1-60761-691-7_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-691-7_9

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-690-0

  • Online ISBN: 978-1-60761-691-7

  • eBook Packages: Springer Protocols