Loss of H1 Impairs Stem Cell Differentiation Figure 3. H1 TKO ESCs fail to undergo neural differentiation. (A) Neural differentiation scheme for ESCs. (B) Characterization of WT and H1 TKO cultures on day 6+7 under neural differentiation protocol. i). Phase contrast images shows that H1 TKO mutants were unable to adequately form neurites and neural networks. Right panels: zoom-in images of the areas encircled with black rectangles. Scale bar: 100 mm (left panels) and 50 mm (right panels). ii). Left panel: Percentage of neurite-forming EBs. Numbers were averaged from 6 experiments. 80 EBs were counted per experiment. Right panel: Numbers of neurites per neurite-forming EB. Number of neurites was counted from EBs that produced neurites. 58 and 28 neurite- forming EBs from respective WT and TKO were selected and counted for neurite numbers. **: P,0.01; ****: P,0.0001. iii). Immunostaining for expression of TUBB3 and GFAP. Nuclei were stained with Hoechst 33342. Scale bars: 50 mm (left panels) and 20 mm (right panels). Results are representative of three independent experiments. (C) H1 TKO ESCs were unable to adequately repress the pluripotency genes and to efficiently induce the expression of neural genes. Expression levels of pluripotency genes (Oct4 and Nanog), neural marker (Nestin), neuronal marker (Tyrosine PLoS Genetics | www.plosgenetics.org 6 May 2012 | Volume 8 | Issue 5 | e1002691