Neurosphere culture derived from aged hippocampal dentate gyrus

The neurosphere assay is the gold standard for determining proliferative and differentiation potential of neural progenitor cells (NPCs) in neurogenesis studies ^1-3 . While several in vitro assays have been developed to model the process of neurogenesis, they have predominantly used embryonic and early postnatal NPCs derived from the dentate gyrus (DG). A limitation of these approaches is that they do not provide insight into adult-born NPCs, which are modeled to affect hippocampal function and diseases later in life. Here, we show a novel free-floating neurosphere culture system using NPCs isolated from the DG of mature adult and aged mice. The protocol outlines detailed steps on the isolation, propagation, and maintenance of neurospheres from adult and aged (>12 months old) mouse brain and how to differentiate cultured neurospheres into neurons and astrocytes. Culturing adult and aged NPCs provides an important in vitro model to (1) investigate cellular and molecular properties of this unique cell population and (2) expand the understanding of plasticity in the adult and aging brain. This protocol requires ∼2 hours to complete dissection, dissociation and culture plating, while differentiation to neuronal and astrocytic lineages takes 9 days. By focusing on neurospheres obtained from animals at later ages this model facilitates investigation of important biological questions related to development and differentiation of hippocampal neurons generated throughout adult life.