Conditional Induction of Math1 Specifies Embryonic Stem Cells to Cerebellar Granule Neuron Lineage and Promotes Differentiation into Mature Granule Neurons§

Authors

  • Rupali Srivastava,

    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
    3. National Brain Research Centre, NBRC, Manesar, India
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  • Manoj Kumar,

    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
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  • Stéphane Peineau,

    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
    3. MRC centre for Synaptic Plasticity, School of Physiology and Pharmacology, Bristol, United Kingdom
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  • Zsolt Csaba,

    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
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  • Shyamala Mani,

    1. National Brain Research Centre, NBRC, Manesar, India
    2. Centre for Neuroscience, IISC, Bangalore, India
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  • Pierre Gressens,

    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
    3. Centre for the Developing Brain, Imperial College, Hammersmith Campus, London, United Kingdom
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  • Vincent El Ghouzzi

    Corresponding author
    1. Inserm, U676, Paris, France
    2. Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France
    • Inserm U676, Hôpital Robert-Debré, 48 Boulevard Sérurier, F-75019 Paris, France
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    • Telephone: +331-40031973; Fax: +331-40031995


  • Author contributions: R.S.: generation, characterization, differentiation of mouse stem cells, real-time PCR, immunocytochemistry, fluorescent microscopy, imaging, cell counting, collection, analysis and interpretation of data, and writing of material and methods section; M.K.: stem cells culture, differentiation, immunocytochemistry, immunohistochemistry, confocal microscopy, and imaging; S.P.: electrophysiological experiments and analyses; Z.C.: in vivo implantations and surgery; S.M.: conception of the study, data interpretation, and manuscript writing; P.G.: data interpretation, financial support, and final approval of manuscript; V.E.G.: conception and design of the study, Western blots, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript. S.M. and P.G. contributed equally to this article.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLS EXPRESS December 7, 2012.

Abstract

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed “T-shaped” axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage. STEM CELLS 2013;31:652–665

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