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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 606 |
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 |
fluorescence-activated
cell sorting; negative selection; positive selection; neural stem cells; lineage-restricted
progenitors;
growth factors; calcium imaging |
coverslips, which were photo-etched
with an alphanumeric grid (Bellco Glass Inc.,
Vineland, NJ) and preglued to 35 mm tissue culture dishes (MatTek
Corp., Ashland, MA).
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Inverted
Fluorescence Microscopy |
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E13
telencephalic |
| Abstract |
| |
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 bFGFcontaining medium,
whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in
bFGF- and EGFcontaining 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-containingmediumrevealed
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. |
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