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Author contributions The following are the supplementary data related to this article.
Acknowledgments Stemformatics is funded by a grant from the Australian Research Council Special Initiative in Stem Cell Science to CAW through Stem Cells Australia. Stemformatics is housed at the Queensland facility for Advanced Bioinformatics (QFAB). We thank Ted Liefield and Michael Reich from the Broad Institute for help with GenePattern integration. We thank Charles Willmore, Willmore designs, Brisbane Australia, for advice on website design and navigation.
Introduction The mammary fat pad in vivo transplant (IVT) assay is widely used for demonstrating multilineage differentiation of murine mammary stem levosimendan (MaSCs). However, this assay is costly, time-consuming, and technically challenging (Stingl, 2009). A less expensive and faster assay for qualifying MaSCs is the in vitro mammosphere assay, in which cells with self-renewal properties, such as stem cells, form spherical structures. This assay was established to identify MaSCs, similar to the neurosphere assay (Dontu et al., 2003). Yet, these assays have been unreliable because of concerns about the clonal origin of the resulting spheres (Deleyrolle et al., 2008; Louis et al., 2008; Reynolds and Rietze, 2005; Singec et al., 2006; Stingl, 2009)
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Discussion The combined findings from time-lapse microscopy and co-culture experiments suggest that most mammospheres formed from basal cells are clonal expansions of stem cells. This does not necessarily indicate a single cell origin because only approximately 50% spheres were initiated from a single cell. This conclusion appears to support the concept that contacts with other cells may provide cues for the stem cells to proliferate and form spheres in suspension culture, in which the natural stem cell niche is absent. This also could be a primary explanation for the low efficiency of sphere formation by single cells. Subsequent sphere differentiation in Matrigel culture showed distinct morphologic differences between 3D structures derived from spheres formed from basal versus luminal cells. These results validate the utility of in vitro sphere differentiation for lineage determination. Furthermore, we directly demonstrated that the single 3D solid structure did contain stem cells that were capable of multilineage differentiation and self-renewal upon transplant in vivo. In concordance with recent findings that MaSCs can be retained by in vitro passage of solid 3D structures in a similar culture system using 5% Matrigel (Guo et al., 2012), our data also suggests that the Matrigel culture system allows MaSC differentiation and retains MaSC self-renewal properties in vitro. The mammosphere assay was initially designed as a serial passage assay to evaluate self-renewal of human mammary stem cells (Dontu et al., 2003). However, its application in detection of mouse mammary stem cells has been deferred due to concerns on sphere aggregation as well as whether all sphere-initiating cells are truly MaSCs (Booth et al., 2007; Liao et al., 2007; Moraes et al., 2007). Our study for the first time revealed that the basal, luminal and stromal cells within the primary mammary cells are all able to form mammospheres when they were cultured alone under non-adherence condition, and stromal cell formed spheres were from cell aggregation. Our finding also indicated that sphere formation efficiency is approximately 7-fold higher by luminal cells than that by basal cells. Therefore, it is expected that spheres derived from primary mammary cells have a mixed pool of cell origins and were dominated by luminal derived spheres as well as merged spheres united by the stromal cells. This may explain why primary mammosphere formation and stem cell number went in opposite directions and why IVT of individual mammospheres formed from the primary unfractionated cells yielded a low (15%) engraftment frequency in a previous study (Moraes et al., 2007).