Eyes Wide Open: A Critical Review of Sphere-Formation as an Assay for Stem Cells

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 an 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.

Understanding cancer stem cell heterogeneity and plasticity

“…CSCs, now reported in most human tumors, are commonly identified and enriched using strategies for identifying normal stem cells, which include flow cytometrybased sorting using cell surface markers, such as CD44 and CD133, and functional approaches, including SP analysis [7], Aldefluor assay [8,48], and sphere formation coupled with serial sphere passaging [49]. The CSCenriched populations prospectively purified using these strategies are then implanted, at various cell doses, in immune-deficient mice to assay their tumor-regenerating capacity, an in vivo assay often called limiting dilution assay.…”

“…Human BCSCs have been enriched in CD44 + CD24 -/lo [50], SP [51,52], ALDH + [53,54], and PKH26 dye-retaining [55] cells. Although the interrelationships between these different subsets of tumorigenic BCSCs remain unclear, these observations [49][50][51][52][53][54][55] suggest that human BCSCs are phenotypically diverse. Indeed, recent studies [53][54][55][56][57][58][59] indicate that not only CSC marker expression in breast cancer cells is heterogeneous but also there exist many subsets of BCSCs that vary from patient to patient, which may be related to individual tumor's genetic makeup [44].…”

“…The stemlike GBM cells have been enriched using cell surface molecules, such as CD133 [61,72], SSEA-1 (stagespecific embryonic antigen-1) [64], EGFR [67,68], and CD44 [69], or functional assays, including the SP analysis [7,63,73] and neurosphere assays [49,74]. Intriguingly, there are significant uncertainties surrounding the use of CD133 as a marker for brain tumor stem cells (BTSCs).…”

“…For example, although LSC activity has been detected in several cellular compartments, the most tumorigenic subpopulation is still in undifferentiated Lin -CD34 + CD38 -cells, which can develop into more mature, less tumorigenic cells [112,113,115]. Likewise, in marker-independent sphere-(e.g., neurosphere, mammosphere, colonosphere, prostasphere, etc) formation assays, it has been repeatedly demonstrated that a single (undifferentiated) tumor cell can generate a sphere that contains multiple types of lineage-differentiated cells [49,61,66,76,80,90,95,100,106]. CD133 + colon CSCs can grow exponentially for more than a year as undifferentiated tumor spheres in vitro but, upon transplantation in vivo, they can generate tumors that histologically recapitulate the morphological and antigenic pattern of the original tumor [76].…”

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

“…Sphere-forming assays are widely used as an in vitro measure of tumor cell self-renewal (24) and have been recently used to assess self-renewal in human ERMS cell lines (25). To assess the effect of activating canonical WNT/ β-catenin pathway on the rhabdosphere forming potential, RD and 381T cells were seeded at limiting dilution and treated with BIO and WNT3A.…”