Sphere-forming assays have been widely used to retrospectively identify stem cells based on their reported capacity to evaluate self-renewal and differentiation at the single-cell level in vitro. The discovery of markers that allow the prospective isolation of stem cells and their progeny from their in vivo niche allows the functional properties of purified populations to be defined. We provide a historical perspective of the evolution of the neurosphere assay and highlight limitations in the use of sphere-forming assays in the context of neurospheres. We discuss theoretical and technical considerations of experimental design and interpretation that surround the use of this assay with any tissue.
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