Figure 5: Loss-of-Oct-4 associated hESC differentiation is mirrored by a dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states. (A) The hESC colony structural zones of spontaneous differentiation. The small compact cells at the core of the colony (Zone 1 [Z-1]) are SSEA-3 (red) and Nestin (green) negative; cells at the periphery (Zone 2 [Z-2], delineated by double-headed white arrows) begin to express SSEA-3 and Nestin; and cells migrated beyond the colony (Zone 3 [Z-3]) continue to express Nestin but lose SSEA-3 expression. Decreased expression of Oct-4 (red) in transitional Zone-2 cells is associated with transiently increased H3 acetylation (acH3, green). Analysis of protein intensities from quantitative intracellular imaging confirms that the decreased expression of Oct-4 in transitional Zone-2 cells is associated with transiently increased histone acetylation, coincident with enhanced expression of p300. (B) Inhibition of HDAC activities results in hESC differentiation. The hESCs maintained under the defined culture were treated with specific HDAC inhibitor trichostatin A (TSA); controls were similarly treated with DMSO. Photomicrographs are shown 3 days after removal of the HDAC inhibitor. Inhibition of HDAC activity resulted in the extinguishing of Oct-4 expression and the appearance of large differentiated Oct-4-negative cells throughout the colony that began to express Nestin (red) and phalloidin (green), and show significantly reduced immune-reactivity to acetylated histone H3 (acH3, green) and acetylated histone H4 (acH4, green). All cells are shown by DAPI staining (blue). White arrows indicate the colony edge.