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Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Nature Biotechnology volume 21, pages 1200–1207 (2003)Cite this article

Abstract

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer–derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into γ-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell–derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell–derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.

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Figure 1: MS5-induced neural differentiation of mouse ES cells.
Figure 2: Astrocytic and oligodendrocytic differentiation.
Figure 3: Neuronal fate specification.
Figure 4: Schematic representation of culture conditions.
Figure 5: Variability of dopamine (DA) neuron differentiation and in vitro function of ES cell–derived neurons.
Figure 6: In vivo function of ES cell–derived neurons.

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Acknowledgements

We thank R. McKay for the Nestin antibody, A. Nagy for the ESB5 cell line, T. Wakayama for the ntES cell lines and E. Lai for the BF1/lacZ ES line. We also thank A. Saxena, P. Song and V. Pratomo for excellent technical assistance, K. Weisel for providing AGM stromal cell lines and R. Stan for a critical review of the manuscript. This work was supported in part by research grants 01.2001.011 and 01.2002.07 from the Michael J. Fox Foundation for Parkinson's Research, and by National Institute of Neurological Disorders and Stroke–National Institutes of Health grant 1R21NS44231-01, as well as by The Parkinson Disease Foundation.

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Authors and Affiliations

  1. Division of Neurosurgery and Developmental Biology Program, Laboratory of Stem Cell and Tumor Biology, Weill Medical College of Cornell University, New York, 10021, New York, USA

    Tiziano Barberi, Hyojin Lee, Kathleen Loonam, Anselme L Perrier, Viviane Tabar & Lorenz Studer

  2. James Ewing Laboratory for Developmental Hematopoiesis, Cell Biology Program, Sloan-Kettering Institute, Weill Medical College of Cornell University, New York, 10021, New York, USA

    Tiziano Barberi & Malcolm A S Moore

  3. Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, 10021, New York, USA

    Peter Klivenyi, Noel Y Calingasan, Hibiki Kawamata & M Flint Beal

  4. Laboratory of Neural Development, Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, 1275 York Avenue, New York, 10021, New York, USA

    Juan Bruses

  5. Department of Physiology and Neurobiology, The University of Connecticut, Storrs, 06269, Connecticut, USA

    Maria E Rubio

  6. Departments of Anesthesiology and Pharmacology, Weill Medical College of Cornell University, New York, 10021, New York, USA

    Norbert Topf & Neil L Harrison

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  1. Tiziano Barberi
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Correspondence to Lorenz Studer.

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Barberi, T., Klivenyi, P., Calingasan, N. et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol 21, 1200–1207 (2003). https://doi.org/10.1038/nbt870

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