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

Nature Protocols volume 3, pages 1888–1894 (2008)Cite this article

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.

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Figure 1: Schematic diagram of differentiation of hESCs into DA neurons.
Figure 2: Morphologies of hESCs and EBs observed during stage 1 of Figure 1.
Figure 3: Morphologies of various neural structures observed during the middle part (NP selection and expansion steps) of the stage 1 of Figure 1.
Figure 4: Morphologies of non-neural (cystic- or spot-forming) structures frequently observed at the initial stage of sphere formation and also during the purification processes.
Figure 5: The size of SNMs before and after fragmentation for either expansion or cryopreservation.
Figure 6: Morphological changes observed during DA neuron generation from the fragmented SNMs.

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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.

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Authors and 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. Myung-Soo Cho
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Correspondence to Dong-Youn Hwang or Dong-Wook Kim.

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Cho, MS., Hwang, DY. & Kim, DW. Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nat Protoc 3, 1888–1894 (2008). https://doi.org/10.1038/nprot.2008.188

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