Observational examination of aggregation and migration during early phase of neurosphere culture of mouse neural stem cells

Neural stem cells (NSCs) form cell aggregates called neurospheres during serum-free culture, and this aggregation contributes primarily to neurosphere development. In the present study, we conducted an observational examination of the cell clustering of mouse NSCs in relation to their migration potential. The cell clustering became significant from 12 h after the inoculation of NSCs during the examined culture period of 48 h. Three types of clustering were observed, that is, aggregation between two single cells, and coalescence between a cell and an aggregate and that between two aggregates. The aggregation between single cells occurred during the early phase (0-24 h after inoculation). Six hours after inoculation, the average migration rate of single cells that resulted in aggregation within the subsequent 12 h was higher than that of cells that did not aggregate. Fluorescent stainings of the cytoplasm and F-actin revealed the existence of mini podia and long podia, which were possible elements that produce the driving force for the migration of singly occurring NSCs.