Abstract
Directed differentiation of human embryonic stem cells (hESCs) to a functional cell type, including neurons, is the foundation for application of hESCs. We describe here a reproducible, chemically defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6–7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural rosettes are enriched by detaching rosettes and leaving the peripheral flat cells attached and expanded as neuroepithelial aggregates in the same medium. Finally, the neuroepithelial aggregates are dissociated and differentiated to nearly pure neurons. This stepwise differentiation protocol results in the generation of primitive neuroepithelia at day 8–10, neural progenitors at the second and third week, and postmitotic neurons at the fourth week, which mirrors the early phase of neural development in a human embryo. Identification of the primitive neuroepithelial cells permits efficient patterning of region-specific progenitors and neuronal subtypes such as midbrain dopaminergic neurons.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zhang SC (2003) Embryonic stem cells for neural replacement therapy: prospects and challenges. J. Hematother. Stem Cell Res. 12, 625–634.
Du ZW, and Zhang SC (2004) Neural differentiation from embryonic stem cells: which way? Stem Cell Development. 13, 372–381.
Zhang SC, Wernig M, Duncan ID, Brüstle O, and Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129–1133.
Pankratz MT, Li XJ, Lavaute TM, Lyons EA, Chen X, Zhang SC. (2007) Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells. 25, 1511–1520.
Hemmati-Brivanlou A, Melton D. (1997) Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell, 88, 13–17.
Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang SC. (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol. 23, 215–221.
Li XJ, Hu BY, Jones SA, Zhang YS, Lavaute T, Du ZW, Zhang SC. (2008) Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells. 26, 886–893.
Yang D, Zhang ZJ, Oldenburg M, Ayala M, Zhang SC. (2008) Human embryonic stem cell-derived dopaminergic neurons reverse functional deficit in parkinsonian rats. Stem Cells. 26, 55–63.
Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC. (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells. 23, 781–790.
Acknowledgments
This study was supported by the National Institute of Neurological Disorders and Stroke (NS045926, NS046587), the Michael J. Fox Foundation, and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2006
About this protocol
Cite this protocol
Zhang, XQ., Zhang, SC. (2009). Differentiation of Neural Precursors and Dopaminergic Neurons from Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 584. Humana Press. https://doi.org/10.1007/978-1-60761-369-5_19
Download citation
DOI: https://doi.org/10.1007/978-1-60761-369-5_19
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-368-8
Online ISBN: 978-1-60761-369-5
eBook Packages: Springer Protocols