The aim of this review is to provide an overview of the fundamental features of the neurosphere assay (NSA), which was initially described in 1992, and has since been used not only to detect the presence of stem cells in embryonic and adult mammalian neural tissues, but also to study their characteristics in vitro. Implicit in this review is a detailed examination of the limitations of the NSA, and how this assay is most accurately and appropriately used. Finally we will point out criteria that should be challenged to design alternative ways to overcome the limits of this assay.

NSA is used to isolate putative neural stem cells (NSCs) from the central nervous system (CNS) and to demonstrate the critical stem cell attributes of proliferation, extensive self-renewal and the ability to give rise to a large number of differentiated and functional progeny. Nevertheless, the capability of neural progenitor cells to form neurospheres precludes its utilisation to accurately quantify bona fide stem cell frequency based simply on neurosphere numbers. New culture conditions are needed to be able to distinguish the activity of progenitor cells from stem cells.

A commonly used, and arguably misused, methodology, the NSA has provided a wealth of information on precursor activity of cells derived from the embryonic through to the aged CNS. Importantly, the NSA has contributed to the demise of the ‘no new neurogenesis’ dogma, and the beginning of a new era of CNS regenerative medicine. Nevertheless, the interpretations arising from the utilisation of the NSA need to take into consideration its limits, so as not to be used beyond its specificity and sensitivity.