The current data indicated that the state of epigenetic marks evolved with differentiation of NSCs. The topographical co-registration of epigenetic modifications and the various differentiation states of differentiating cells in zoning in and out of neurospheres allowed us, in part, to discern the epigenetic program of differentiating NSCs (Figure 2). During the four-day culture, some NSCs remained undifferentiated in the core of neurospheres; others began, or were about to begin, the differentiation process in periphery of the neurosphere and advanced into differentiation as they migrated away from the neurosphere. In parallel, the histone marks that remained highly prevalent in the neurosphere core were greatly reduced in the periphery zone of neurospheres, and largely diminished in the migrated NSCs. This temporal and spatial correlation suggests that turning off the high profile Ac-H4, 2me-H3K4, and 3me-H3K27 marks in association with the previously engaged genes for stem cell maintenance is a prerequisite of the NSC restriction into neural differentiation. This is demonstrated by migrated cells that: contained diminished histone marks, OCT4-negative immunostaining, and acquired neural morphology with neural fibers and smooth outlines, all
