The floating neurospheres obtained from the cultures remained morphologically undifferentiated: cells were round and small and had a distinct single nucleus in the center of the cytoplasm. Many cells in the undifferentiated neurosphere (Figure 1A,C,E) were OCT4+ with a small fraction also expressing Sox2+, indicating a mixed heterogeneous population with various advanced stem cell status. Low expression of glial fibrillary acidic protein (GFAP) was seen in undifferentiated neurospheres. After four-days culture on substrate-coated chamber slides, the NSCs demonstrated a transition of differentiation and migration out from the center of the neurosphere as reported previously (22). In the core of the differentiating neurosphere, the NSCs were round and small, OCT4+ (Figure 1B), nestin-negative, and dividing cells were noted by immunostaining (-im) for PCNA (not shown). Dividing nuclei were also occasionally present. In the periphery of the neurosphere, much like the undifferentiated groups, many NSCs remained round, but a subpopulation of the cells formed angular cytoplasmic expansions, some with fiber extensions. In the migrated zone, more differentiated cells migrated out of neurosphere, some retaining Sox2 expression (Figure 1D) and acquired glial or
