A dose–response curve of hNGN2-induced neurons treated with Ara-C from days 6–20 of our protocol demonstrated that low concentrations (10 nM) of Ara-C was insufficient to eliminate Ki67-positive replicative cells in our culture, while substantial cell death was observed in cells treated with Ara-C concentrations of 1 μM (Fig. 4F). We observed robust reduction of replicative cells across all NPC lines tested with 50–100 nM Ara-C treatment from days 6–20 (more than 5-fold, p < 0.05) (Fig. 4G and H). Thus, our recommendation is to include 50 nM Ara-C whenever experimental design requires excluding proliferative cells, although it is may be possible to reduce the length of Ara-C treatment.
