Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Abstract

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass1,2 or developing germ cells3 can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal4,5,6,7. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.

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Figure 1: ES cells progressively differentiate into mesencephalic stem cells.
Figure 2: Mesencephalic precursors differentiate into TH+ neurons.
Figure 3: The effect of external signals on the yield of neurons that are TH+ and secrete dopamine.
Figure 4: Synaptic properties of ES-derived TH+ neurons.
Figure 5: Characterization of serotonin-positive neurons.

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Acknowledgements

The R1 ES cell line was kindly provided by Dr. Heiner Westphal. The second ES cell line used E14.1 was a gift of Dr. Tom Doetschman. We would like to thank Dr. David Panchinsion for providing some of the PCR-primers and for critical discussions of the manuscript.

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Correspondence to Ron D. McKay.

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Lee, S., Lumelsky, N., Studer, L. et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 18, 675–679 (2000). https://doi.org/10.1038/76536

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