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. 2011 May 6;8(5):486-98.
doi: 10.1016/j.stem.2011.04.007.

Eyes wide open: a critical review of sphere-formation as an assay for stem cells

Erika Pastrana  1 , Violeta Silva-Vargas, Fiona Doetsch
Affiliations

Eyes wide open: a critical review of sphere-formation as an assay for stem cells

Erika Pastrana et al. Cell Stem Cell. .

Abstract

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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Figures

Figure 1
Figure 1. Schema of the neurosphere assay
Figure 2
Figure 2. SVZ anatomy and lineage
A, Frontal schema of adult mouse brain showing SVZ in orange adjacent to the lateral ventricle. B, Summary schema of the organization of SVZ cells. GFAP+ stem cells (B, blue) transit amplifying cells (C, green), neuroblasts (A cells, red), are adjacent to ependymal cells (E, grey), which line the lateral ventricle (LV). A subset of GFAP+ cells (B1) extend a process between ependymal cells to contact the LV. Stem cells and transit amplifying cells directly contact the vasculature at specialized regions on blood vessels lacking astrocyte endfeet (dark blue) and pericyte coverage (yellow). C, Confocal image of ependymal cell pinwheel with GFAP::GFP+ Type B1 cell contacting the ventricle at its center (Image from Angel Maldonado-Soto, generated according to the methods reported in Mirzadeh et al., 2008). D, Stem cell lineage and sagittal schema of adult mouse brain showing whole mount perspective of SVZ adjacent to lateral ventricle (red). Newly generated neurons migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB). Small numbers of oligodendrocytes are also generated in the SVZ but are not shown here. Beige boc indicates neurosphere forming cells in the lineage.
Figure 3
Figure 3
Flowchart outlining design and critical experimental steps in sphere-forming assays
See this image and copyright information in PMC

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