Table 3.

Overview of critical considerations for sphere-forming assays

Cell density and clonality of spheres	Cell density is critical parameter as it influences clonality. Spheres are prone to aggregation due to both intrinsic locomotion and to experimenter-induced movement. Clonality is only guaranteed by single cell plating. Important to ensure that spheres are due to proliferation of cells and not to aggregation.
Sphere-forming assays may not detect quiescent stem cells	Sphere forming assays predominantly detect cells that are poised for, or are actively undergoing, proliferation. Quiescent cells may not be capable of forming spheres, either due to intrinsic cell properties or due to lack of additional extrinsic signals needed for their activation in this assay.
Sphere-forming assays are not a readout of in vivo stem cell frequency	Multiple populations in stem cell lineages, including both stem cells and transit amplifying cells, are able to form spheres that can be serially passaged and are multipotent. The long-term in vitro and in vivo potential of purified populations needs to be assessed.
Sphere size is not a read-out of in vivo stem cells	Large spheres are often assumed to arise from stem cells. However, independent of aggregation issues, sphere size simply reflects proliferation/ differentiation status and responsiveness to growth factors of the parental clone-forming cell.
Towards the prospective purification of sphere-forming cells	As FACS becomes an integral part of assaying the potential of different populations to form spheres, it is essential to ensure that enzymatic digestion does not alter surface marker profiles (different enzymes result in markedly different surface profiles). In addition, the cell type specificity of individual markers needs to be validated in vivo.
Markers are dynamic	Purified populations reflect the state of a population at a given moment in time. Within a purified population, cells may be in different states or stages of the cell cycle. Markers commonly used to purify stem cells change their expression with the cell cycle. Cells can shuttle between quiescent and activated states, or from more committed to more primitive states.
Differentiation potential bias due to culture with exogenous growth factors	Cells within a population may respond differently to distinct mitogens. Mitogens can also bias the differentiation capacity of cells.