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.