Neural stem cells (NSCs) can be cultured as floating neurospheres in vitro. However, it remains unclear whether these clusters of several hundreds or even thousands of cells emerge from a single proliferating cell or from an assembly of cells. To answer this question, we monitored neurosphere re-formation after passaging of cultures. We isolated NSCs by microdissection of the subventricular zone of C57BL/6 mice and established cultures that were passaged every 7 days according to standard protocols. We used time-lapse video microscopy to observe cell behavior 24, 48 or 72 h after passaging for an interval of 18 h. We found strong variations in the morphology and growing patterns early after each passage. Single cells attached to the culture dish to form layer-like networks or remained floating. The floating cells formed small neurospheres (24 h) that constantly expanded by proliferation over the following hours. Adherent cells formed networks (24 h), followed by migration and aggregation (48 h). The aggregates finally detatched, giving rise to floating neurospheres (72 h). These findings demonstrate that neurospheres are not generated from one "founder" cell but must be regarded as a multi-clonal final assembly of cells which started from the aggregation of individual cells.

Support: German Research Foundation (DFG, MA 2492/2-2).