HDACi greatly enhance hPSCs differentiation towards NPCs. (A) Schematic representation of the neural differentiation in monolayer culture. (B) Immunostaining of cells derived from neurospheres with antibodies against NESTIN (green) and SOX2 (red) and quantification of NESTIN and SOX2 dual positive cells on day 18 of differentiation. The nuclei were stained by Hoechst. Scale bar, 50 μm. The inset shows a higher magnification view. Scale bar, 100 μm. (C) Immunostaining of cells after 40 days of spontaneous differentiation with antibodies against MAP2 (green) and GFAP (red). The nuclei were stained by Hoechst. Scale bar, 50 μm. The inset shows a higher magnification view. Scale bar, 100 μm. (D) Neurospheres were formed after HDACi treatment for seven days. The sizes of neurospheres were analyzed on day 18 of differentiation. Black arrows represent typical neurospheres, and red arrowheads represent cell debris. (E-G) Neurospheres were formed after MGCD treatment for seven days and the diameters of the neurospheres were measured and quantified on day 18, respectively. (H-J) Neurospheres were formed after NaB treatment for seven days or eighteen days and the diameters of the neurospheres were detected and quantified on day 18 of differentiation, respectively. Scale bars, 200 μm. The error bars indicate SEM; ns, not significant; ***P <0.001; n >50. (K) The efficiency of NPC generation was assessed by counting the number of cells derived from digested neurospheres on day 18. **P <0.01; n >/=3. HDACi, HDAC inhibitors; hPSCs, human pluripotent stem cells; NPC, neural progenitor cell; SEM, standard error of the mean.