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Fig. 1. Expansion of primary neural cells as a homogenous population of
AHNPs. (A) Highly expanded (more than 60 PDs) cells ubiquitously
express nestin (red), with a large subset of GFAP+ cells (green).
(B) AHNPs express widespread immature neuronal and glial markers,
including A2B5 (red) and NG2 (green). (C,D) AHNPs
(nestin+, green) express astrotypic markers in a large subset of
cells, including S100ß (C, red) and glutamine synthetase (D, green).
(E) Voltage-clamp profile of these cells reveal prominent
Na+ and K+ channel activity. Data shown for temporal
cortex-derived cells. (F) Nestin+ (green) AHNPs proliferated
in the presence of BrdU (red) uniformly incorporate thymidine analog.
(G) Stereological evaluation of proliferating AHNPs reveals a uniform
nestin+ population that frequently co-expresses glial cell markers
(GFAP shown). Maintaining these cells in growth medium supplemented with BrdU
results in label saturation in AHNPs (BrdU+Nestin+
cells) at a rate of incorporation analagous to previously characterized
proliferative dynamics (H). Removal of mitogenic stimuli (GF=EGF+bFGF)
results in failure of AHNPs to divide (see
Fig. 2F). (I,J)
Both hippocampal and temporal cortex-derived AHNPs maintain comparable stable
doubling rates and uniform protoplasmic morphologies throughout culture.
(K) AHNPs derived from temporal cortex and hippocampus reveals
continuous logarithmic expansion throughout culture. Scale bars: 25 µm in
A,B,F,J; 50 µm in C; 75 µm in D. Images counterstained with DAPI.
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