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Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen

Nature Protocols volume 6, pages 1229–1240 (2011)Cite this article

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Abstract

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O2) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O2, where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at 20% O2, but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of fibroblast growth factor-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription factor 2) induction in motor neuron precursors is twofold greater than that at 20% O2, whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O2) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O2 should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.

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Figure 1: hESC culture.
Figure 2: Procedure for neural conversion.
Figure 3: NPC generation at 3% O2.
Figure 4: Functional differentiation at 3% O2.
Figure 5: Spinal motor neurons.
Figure 6: Midbrain dopaminergic neurons.

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Acknowledgements

We are grateful to L. Vallier for kindly providing feeder-free ES cells, M. Alexander for valuable technical assistance and R. Barker for the use of the low-oxygen incubator. This work was supported by the Multiple Sclerosis Society UK, the Evelyn Trust, the Medical Research Council, the National Institute for Health Research (Cambridge Biomedical Research Centre), the Wellcome Trust (A.L.) and the Royal Society (R.K.). S.R.L.S. is supported by a Sir David Walker Fellowship, a joint Medical Research Council and Multiple Sclerosis Society Clinical Research Training Fellowship (no. G0800487) and a Raymond and Beverly Sackler Studentship. We obtained the hybridomas Hb9 (MNR2) and En1, developed by T.M. Jessell and S. Brenner-Morton, from the Developmental Studies Hybridoma bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biology at the University of Iowa.

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  1. S R L Stacpoole and B Bilican: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK

    S R L Stacpoole, D J Webber, X L He & A Compston

  2. MRC Centre for Stem Cell Biology and Regenerative Medicine and Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

    S R L Stacpoole, A Luzhynskaya, R Karadottir & R J M Franklin

  3. Euan MacDonald Centre and Centre for Neuroregeneration, University of Edinburgh, Edinburgh, UK

    B Bilican & S Chandran

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Contributions

S.R.L.S., B.B., D.J.W. and S.C. designed the experiments; S.R.L.S., B.B. and X.L.H. conducted the experiments; A.L. and R.K. performed the neuronal electrophysiology; S.R.L.S., B.B., A.L. and R.K. analyzed the data; A.C., R.J.M.F. and S.C. supervised the project; S.R.L.S., B.B., D.J.W., A.L., A.C., R.K., R.J.M.F. and S.C. wrote the protocol.

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Correspondence to S R L Stacpoole.

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Stacpoole, S., Bilican, B., Webber, D. et al. Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen. Nat Protoc 6, 1229–1240 (2011). https://doi.org/10.1038/nprot.2011.380

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