Since the sizes of neurospheres were not all identical, the migration density of cells (i.e. number of differentiated cells per neurosphere) would be affected by this variable. We thus did not analyze the epigenetic marks by cell number but rather the neurosphere was divided into three regions: core, corresponding to the densely packed inner mass of tightly clustered cells; periphery, a zone stretching out approximately 100μm from the edge of the core, consisting of early differentiating cells that were less densely packed, starting to leave the neurosphere, changing morphologically from round neuroepithelial cells to tear-drop shaped neural-like cells, and some at the outer edge of the periphery with extended neurites; and the migrated zone, consisting of all cells beyond the periphery, which have varying degrees of differentiation. Furthermore, since all cells in the neurosphere were not differentiated equally, the intensity of the epigenetic marks and phenotype expression were individually different, which are hypothesized to correlate with their differentiation status. Thus, it is reasonable that we analyze the population of cell intensity, particularly along the axis of their migration path.
