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Adult neural stem cells (NSC) generate neurons throughout life, but little is known about the sequence of events involved in the transition from NSC to neurons. Studying the intermediary steps involved in the specification of neuronal cells from NSCs requires observation of cells in real time. Here we describe a primary culture of the adult subependymal zone (SEZ) which allows for continuous live imaging to characterize the mode of cell division and lineage progression of adult NSCs and their progeny. To this end, cells are cultured at low density under adherent conditions and without growth factors. Under these conditions, NSCs display classical hallmarks of adult SEZ NSCs in vivo, such as astroglial marker expression and promoter activity, a slow cell cycle, and a predominantly neurogenic potential. Video time-lapse microscopy experiments using this cell preparation allow for studying the steps involved in the generation of fast-dividing precursors and neuroblasts from slow-dividing astroglia/NSCs.

Direct conversion of glia into neurons by cellular reprogramming represents a novel approach toward a cell-based therapy of neurodegenerative processes. Here we describe a protocol that allows for the direct and efficient in vitro reprogramming of mouse astroglia from the early postnatal neocortex by forced expression of single neurogenic fate determinants. By selective retrovirus-mediated expression of neurogenin-2 (Neurog2) on the one hand, or the mouse homologue of Distal-less Dlx2 or the mammalian homologue of achaete-schute-1 (Mash1) on the other, it is possible to drive postnatal astroglia in culture toward the genesis of fully functional, synapse-forming, glutamatergic, i.e., excitatory, and GABAergic, i.e., inhibitory, neurons, respectively.

Neurosphere-forming cells can be isolated from virtually all neural tissues during embryonic development as well as some neural tissues in the adult such as the adult subependymal zone (SEZ) and spinal cord. While these cells share in common the ability of generating clonal aggregates, the so-called neurospheres, that display self-renewal capacity and cellular multipotency (i.e., the potential to generate the three major neural lineages), they can markedly differ in their intrinsic propensity toward neurogenesis or gliogenesis as well as their ability to generate distinct types of neurons with respect to their transmitter identity. Here we discuss the endogenous differentiation potential of neurosphere cells derived from both embryonic and adult brain tissues and provide protocols how to direct adult SEZ neurosphere cells toward specific neuronal subtypes (i.e., GABAergic versus glutamatergic) via retroviral-mediated gene transfer of proneural genes.