Within 3-5 days of FGF-2 removal, neurite-like processes appeared (Fig. 7A). By 1012 days, extensive fibers emanated from clumps of plated NPCs or from spherical structures (Fig. 7B). Networks of fiber bundles connecting cell clusters or spherical structures were also apparent (Fig. 7C). Approximately 45% ± 5% (801/1780, two replicates) of cells stained positive for markers of mature neurons, MAP-2C or NeuN (Fig. 6, C-E). However, in cell clusters or spherical structures with fiber bundles, approximately 69% ± 3% of cells stained positive for MAP-2C (315/459, five replicate) or NeuN. Interestingly, in other areas on the same coverslip, fewer cells were MAP-2C- or NeuN-pos-itive (data not shown), suggesting that neuronal differentiation induced by this protocol may involve random or local development. buy glucophage online
However, the population of MAP-2C-positive cells (83% ± 7%, 916/1108, three replicates) or NeuN-positive cells (76% ± 6%, 822/1082, three replicates) increased after selection of NPC-containing tubular structures for culture after FGF-2 removal. At least some of these neuronal cells (NeuN-positive cells) expressed biochemical markers, including TH and ChAT. The number of double-labeled NeuN and TH cells (Fig. 6D) amounted to less than 4% (18/541, three replicates) of the ES cell-derived neuronal population, whereas almost 13% (73/583, three replicates) of NeuN-positive cells were also ChAT-positive (Fig. 5E). Approximately 29% ± 4% (514/1766, three replicates) of cells in the NPC population expressed the astrocyte-specific antigen GFAP (Fig. 6F), indicating that these cells had differentiated into mature glial phenotypes.
FIG. 6. Immunocytochemical characterization of monkey ES cell-derived NPCs differentiated into neurons and glial cells. The ES cell-derived NPCs immunoreactive for the NPC markers nestin (A, green label) and musashil (B, green label) are shown. Differentiated cells expressing the neuronal-specific protein MAP2 (C, green label), NeuN (E and F, red label), and the glial cell marker GFAP (D, green label) are also shown. Cells in A-D were labeled with the DNA-specific fluorochrome DAPI. The NeuN-positive cells (red label confined to the nucleus) were also examined for the presence of ChAT (E, green label) and TH (F, green label) for specific neuron-expressed neurotransmitter-synthesizing enzymes. Original magnification X250 (A-D) and X400 (E and F).
FIG. 7. Phase-contrast micrographs depicting the differentiation of ES cell-derived NPCs. A) Cells with short process emanating from a spherical structure 3 days after withdrawing FGF-2. B) Cells with long, multiple processes 10 days after withdrawing bFGF. C) A bundle of processes connecting clumps of differentiating cells. Original magnification X100 (A and B) and X200 (C).