Table 3. Percentage of cells expressing neuronal markers during differentiation.
| Day 7 | Day 10 | Day 21 | Day 30 | |
|---|---|---|---|---|
| Nestin | 65 ± 11 | NA | NA | NA |
| Nurr1 | 23 ± 8 | NA | NA | NA |
| NF200 | 0 | 62 ± 7 | 90 ± 5 | 92 ± 6 |
| GFAP | 0 | 15 ± 5 | 3 ± 2 | 2 ± 2 |
| MBP | 0 | 11 ± 3 | 2 ± 2 | 2 ± 3 |
| DDC/TH/Dopa | 0 | 0 | 25 ± 7 | 22 ± 8 |
| TrH | 0 | 0 | 18 ± 3 | 23 ± 2 |
| GABA | 0 | 0 | 52 ± 5 | 52 ± 3 |
Cells were fixed with 4% paraformaldehyde (Sigma) for 4 min at room temperature followed by methanol (Sigma) for 2 min at − 20°C. For nuclear ligands, cells were permeabilized with 0.1 M Triton X-100 (Sigma) for 10 min. Slides were incubated sequentially for 30 min each with primary antibody and FITC or Cy3- or Cy5-coupled anti-mouse-, goat-, or rabbit-IgG antibodies. Between each step, slides were washed with PBS plus 1% BSA (Sigma). Cells were examined by confocal fluorescence microscopy (confocal 1024 microscope, Olympus AX70). To assess the frequency of different cell types in a given culture, we counted the number of cells staining positive with a given antibody in four visual fields (50-200 cells per field). Results shown are mean ± SEM from three independent differentiations from MAPCs to neuroectodermal cells evaluated after 7-30 days.