Loss of H1 Impairs Stem Cell Differentiation Figure 1. Loss of H1c/H1d/H1e inhibits spontaneous ESC differentiation. (A) Western blot analysis of OCT4 level in WT and H1 TKO ESCs cultured under indicated conditions for 2 days. (B) Phase images of WT and H1 TKO ESCs cultured either on MEF with LIF (left panel), gelatin coated plate with LIF (middle panel), or gelatin coated plate without LIF (right panel) for 2 days. Scale bar: 100 mm. (C) Growth curves of WT and H1 TKO ESCs cultured on gelatin coated plate with or without LIF. Data are presented as average 6 S.D. doi:10.1371/journal.pgen.1002691.g001 tency genes as well as important developmental genes for transcription factors and signaling molecules for all three germ layers. WT and TKO cultures at day 0 displayed very few differences in gene expression and their gene expression profiles clustered most similarly in hierarchical cluster analysis (Figure 2C, 2Di, and Figure S2Bi). WT EBs differentiated as expected with significant increases of many differentiation markers and de- creased expression of pluripotency associated genes (Figure 2C, PLoS Genetics | www.plosgenetics.org 3 May 2012 | Volume 8 | Issue 5 | e1002691