A fast and simple differentiation protocol to study the pro-neurogenic activity of soluble factors in neurospheres

• Neuronal differentiation in SVZ neurospheres occurs within hours.
• Assay to measure differentiation largely independent of cell death and cell proliferation.
• Neuronal differentiation in SVZ neurospheres drops with increasing passage number.
• Inhibition of EGFR-, Erk1/2-, Akt-, and Shh-signaling induces neurogenesis.

Sphere-forming assays are widely used for the propagation, characterization and manipulation of adult brain-derived stem- and progenitor cells. However despite the broad application of this cell culture system in neural stem cell- and brain tumor research, no standardized protocols exist. Variations in experimental procedures not only concern the use of media components but also cell density, the number of passages the cells are propagated before analysis and, in cases where the neurogenic or gliogenic potential of the cells is investigated, the duration that the cells are allowed to differentiate. The latter deserves consideration because the proportion of differentiated cells obtained at the endpoint of the experiment depends not only on the absolute number of cells that differentiate at a given time, but also on the number of cell divisions prior to differentiation and the rate of cell death in the cultures. In the present study we describe a fast and simple differentiation protocol to investigate the pro-neurogenic potential of soluble factors added to subventricular zone (SVZ)-derived neurospheres. The assay relies on the use of primary neurospheres and very short differentiation times, thereby largely excluding the contribution of cell proliferation and cell death to the results. We use this modified assay to test the consequence of pharmacological inhibition of the EGF receptor-, Erk1/2-, Protein Kinase B/AKT-, and Sonic Hedgehog-pathways on neuronal differentiation of SVZ-neurosphere cultures.