Mab5326

Dissociated fourth‐passage HSVtk‐hiPSC‐NS/PCs were plated in poly‐l‐ornithine/fibronectin‐coated 8‐well chamber slides (Thermo Fisher Scientific) at a density of 8.0 × 10⁴ cells per milliliter and cultured in medium without growth factors at 37°C under 5% CO₂ and 95% air for 28 days in total. Four sets were prepared for analysis. Cells in the chambers of two of the four sets were treated with 2 μg/ml DOX and 3 μg/ml GCV during the final 7 days (GCV[+]). The other two sets were treated only with 2 μg/ml DOX (GCV[−]). Differentiated cells were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate‐buffered saline (PBS) and stained with the following primary antibodies: anti‐Nestin (mouse immunoglobulin G [IgG], 1:200; Merck KGaA, MAB5326), anti‐Ki67 (rabbit IgG, 1:200; Abcam, Cambridge, U.K., ab15580), and anti‐β‐III Tubulin (mouse IgG2b, 1:300; Sigma–Aldrich, St. Louis, MO, T8660). Nuclei were stained with Hoechst 33258 (10 μg/ml; Sigma–Aldrich). All in vitro images were obtained using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Jena, Germany). One hundred cells stained with Hoechst 33258 were randomly counted from each well, and Nestin‐, Ki67‐, and β‐III Tubulin‐positive cells were counted.