Isolation and Culture of Adult Hippocampal Precursor Cells as Free-Floating Neurospheres

The neurosphere assay is the most widely used in vitro tool to determine the proliferative and differentiation potential of adult neural precursor cells in rodents. Although originally developed for, and predominantly applied to, the growth of embryonic and adult subventricular zone-derived stem cells, hippocampal neurospheres are now routinely cultured by many laboratories. As hippocampal neurospheres are fewer in number, on average smaller in size, and more slowly growing than their ventricular counterparts, the methodology traditionally used to isolate and culture neurospheres from the subventricular zone is not optimal for hippocampal neurosphere growth. Here, we provide a detailed description of an optimized protocol for the microdissection, dissociation, and neurosphere generation from adult hippocampal dentate gyrus tissue. We also outline the protocols required to perform downstream passaging, differentiation, and immunohistological determination of the multipotentiality of hippocampal neurospheres.