(A) design of time course experiments. Dual SMAD inhibitor induction was carried out from Day 0–7 and cells were assayed for PAX6 expression on Day 16; ID-8 was added from Day 0–16, Day 0–7, Day 0–9, or Day 9–16. (B) phase contrast micrographs showing morphology of control hESC, neural progenitors induced by dual SMAD inhibition, and neural progenitors incubated with 0.5 µM ID-8 throughout the neural induction protocol, on Day 16. (C) dose response study of the inhibition of induction of PAX 6 positive cells by ID-8 showing flow cytometry profile (left panel) and percentage of PAX6 positive cells (right panel). Error bars, SD; *p<0.05, **p<0.0.01. (D) effect of timing of ID-8 exposure at 0.5 µM on inhibition of PAX6 induction. Error bars, SD; **p<0.05, *p<0.0.01. (E) indirect immunofluorescence analysis of NESTIN (green) and POU5F1 expression (red) in control hESC, neural progenitors, and cultures subjected to neural induction in the presence of 5.0 µM ID-8. Nuclear counterstain, dark blue. (F) flow cytometry profiles showing expression of stem cell surface molecules GCMT-2 and CD9 in control cells, neural progenitors and cultures subjected to neural induction in the presence of 5.0 µM ID-8. A-D, studies carried out with HES3 (PAX6mCherry) cell line; E-F, WA09 hESC.