Dopaminergic Differentiation of Human Embryonic Stem Cells

Authors

  • Xianmin Zeng Ph.D.,

    Corresponding author
    1. Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
    • Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 333 Cassell Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565; Fax: 410-550-1621
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  • Jingli Cai,

    1. Laboratory of Neuroscience, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Jia Chen,

    1. Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Yongquan Luo,

    1. Laboratory of Neuroscience, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Zhi-Bing You,

    1. Behavioral Neuroscience Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Erin Fotter,

    1. Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Yun Wang,

    1. Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Brandon Harvey,

    1. Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Takumi Miura,

    1. Laboratory of Neuroscience, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Cristina Backman,

    1. Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Guann-Juh Chen,

    1. Molecular Neuropsychiatry Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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  • Mahendra S. Rao,

    1. Laboratory of Neuroscience, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA
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  • William J. Freed

    1. Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA
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Abstract

In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum.

TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-β-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+.

When transplanted into 6-hydroxydopamine–treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

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