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The Journal of Neuroscience, January 1, 2003, 23(1):240-251

Prospective Cell Sorting of Embryonic Rat Neural Stem Cells and Neuronal and Glial Progenitors Reveals Selective Effects of Basic Fibroblast Growth Factor and Epidermal Growth Factor on Self-Renewal and Differentiation

Dragan Maric, Irina Maric, Yoong Hee Chang, and Jeffery L. Barker

Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the embryonic rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast growth factor receptor type 1 (FGFR-1), and a minority exhibited basic fibroblast growth factor (bFGF), whereas few expressed epidermal growth factor receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and differentiation into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-1⁺, with few EGFR⁺, and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca²⁺ imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca²⁺ (Ca²⁺_c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca²⁺_c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two growth factors permit the initial commitment of neural stem cells into neuronal and glial phenotypes.

Key words: fluorescence-activated cell sorting; negative selection; positive selection; neural stem cells; lineage-restricted progenitors; growth factors; calcium imaging

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Copyright © 2003 Society for Neuroscience  0270-6474/03/231240-12$05.00/0

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