Protocol | Published:

Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale

Nature Protocols volume 3, pages 18881894 (2008) | Download Citation

Subjects

Abstract

Cell-replacement therapy using human embryonic stem cells (hESCs) holds great promise in treating Parkinson's disease. We have recently reported a highly efficient method to generate functional dopaminergic (DA) neurons from hESCs. Our method includes a unique step, the formation of spherical neural masses (SNMs), and offers the highest yield of DA neurons ever achieved so far. In this report, we describe our method step by step, covering not only how to differentiate hESCs into DA neurons at a high yield, but also how to amplify, freeze and thaw the SNMs, which are the key structures that make our protocol unique and advantageous. Although the whole process of generation of DA neurons from hESCs takes about 2 months, only 14 d are needed to derive DA neurons from the SNMs.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Understanding Parkinson's disease. Sci. Am. 276, 52–59 (1997).

  2. 2.

    & Levodopa motor complications in Parkinson's disease. Ann. Neurol. 47, 167–178 (2000).

  3. 3.

    et al. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 24, 1433–1440 (2006).

  4. 4.

    , , , & Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells 26, 55–63 (2008).

  5. 5.

    et al. In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J. Neurochem. 92, 1265–1276 (2005).

  6. 6.

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

  7. 7.

    et al. Neural conversion of ES cells by an inductive activity on human amniotic membrane matrix. Proc. Natl. Acad. Sci. USA 103, 9554–9559 (2006).

  8. 8.

    et al. Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin. Stem Cells 25, 411–418 (2007).

  9. 9.

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

  10. 10.

    et al. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 105, 3392–3397 (2008).

  11. 11.

    et al. Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem Cells 24, 557–567 (2006).

  12. 12.

    et al. Dopamine neurons derived from embryonic stem cells efficiently induce behavioral recovery in a Parkinsonian rat model. Biochem. Biophys. Res. Commun. 341, 6–12 (2006).

  13. 13.

    , , , & Derivation of functional dopamine neurons from embryonic stem cells. Cell Transplant. 16, 117–123 (2007).

  14. 14.

    & Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).

  15. 15.

    et al. Derivation and characterization of new human embryonic stem cell lines, SNUhES1, SNUhES2 and SNUhES3. Stem Cells 23, 211–219 (2005).

  16. 16.

    et al. Methods for expansion of human embryonic stem cells. Stem Cells 23, 605–609 (2005).

  17. 17.

    et al. Efficient induction of oligodendrocytes from human embryonic stem cells. Stem Cells 25, 419–424 (2007).

Download references

Acknowledgements

This research was supported by grants (codes: SC3150, SC5170 and SC1110) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea.

Author information

Affiliations

  1. Stem Cell Research Center, 21C Frontier R&D Program of Ministry of Education, Science and Technology, Yonsei University Medical Centre, 134 Sinchon-dong, Seodaemun-gu, Seoul 120-752, Korea.

    • Myung-Soo Cho
    • , Dong-Youn Hwang
    •  & Dong-Wook Kim
  2. R&D Center, Jeil Pharmaceutical Co. Ltd, 117-1, Keunkog-ri, Baegam-myun, Cheoin-gu, Yongin 449-861, Kyunggi-do, Korea.

    • Myung-Soo Cho
  3. CHA Stem Cell Institute, Pochon CHA University College of Medicine, 606-16 Yeoksam-dong, Kangnam-gu, Seoul 135-081, Korea.

    • Dong-Youn Hwang
  4. Department of Physiology, Yonsei University College of Medicine, 134 Sinchon-dong, Seodaemun-gu, Seoul 120-752, Korea.

    • Dong-Wook Kim

Authors

  1. Search for Myung-Soo Cho in:

  2. Search for Dong-Youn Hwang in:

  3. Search for Dong-Wook Kim in:

Corresponding authors

Correspondence to Dong-Youn Hwang or Dong-Wook Kim.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2008.188

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.