Abstract
Midbrain dopaminergic (mDA) neurons play a critical role in regulating postural reflexes and movement as well as modulating psychological processes. Dysfunction or degeneration of mDA neurons is involved in a number of neurological disorders including Parkinson’s disease. Availability of large quantities of human mDA neurons would greatly enhance our ability to reveal pathological processes underlying mDA neuron degeneration and to identify treatments for these neurological conditions. Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an unlimited source for mDA neurons. Here we describe a chemically defined protocol for mDA neuron differentiation. PSCs are first converted to neuroepithelia in a chemically defined medium without any growth factors, followed by patterning the neuroepithelia to midbrain progenitors with fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH) and subsequent differentiating to functional mDA neurons. This protocol typically yields about half of the neuronal population being mDA neurons, determined by expression of mDA markers, electrophysiological recordings, and the ability to reverse functional deficit in a rat model of Parkinson’s disease.
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References
Smidt MP, Burbach JP. (2007) How to make a mesodiencephalic dopaminergic neuron Nat Rev Neurosci 8, 21–32.
Ye W, Shimamura K, Rubenstein JL, Hynes MA, Rosenthal A. (1998) FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate Cell 93, 755–66.
Barberi T, Klivenyi P, Calingasan NY, et al.(2003) Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice Nat Biotechnol 21, 1200–7.
Perrier AL, Tabar V, Barberi T, et al.(2004) Derivation of midbrain dopamine neurons from human embryonic stem cells Proc Natl Acad Sci USA 101, 12543–8.
Buytaert-Hoefen KA, Alvarez E, Freed CR.(2004) Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF Stem Cells 22, 669–74.
Roy NS, Cleren C, Singh SK, Yang L, Beal MF, Goldman SA.(2006) Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes Nat Med 12, 1259–68.
Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA.(2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 19, 1129–33.
Li XJ, Du ZW, Zarnowska ED, et al.(2005) Specification of motoneurons from human embryonic stem cells Nat Biotechnol 23, 215–21.
Yan Y, Yang D, Zarnowska ED, et al.(2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells Stem Cells 23, 781–90.
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.
Hu, B.Y., Zhang S.C.(2009) Differentiation of spinal motor neurons from pluripotent human stem cells. Nature Protocols 9, 1295–304.
Xia, X.F., Zhang, S.C. (2009) Differentiation of neuroepithelia from human embryonic stem cells. Methods in Molecular Biology. 549, 51–8 (Human Press, New York)
Acknowledgments
This study was supported by the National Institute of Neurological Diseases and Stroke (NS045926, NS046587 to SCZ), and in part by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352), the Ministry of Science and Technology, China (2006CB94700, 2006AA02A101), and the Shanghai Municipality (06dj14001).
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Ma, L., Liu, Y., Zhang, SC. (2011). Directed Differentiation of Dopamine Neurons from Human Pluripotent Stem Cells. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_30
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DOI: https://doi.org/10.1007/978-1-61779-201-4_30
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Online ISBN: 978-1-61779-201-4
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