Abstract
For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.
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Acknowledgements
We acknowledge the contributions of M. Ditter, C.E. Hagemeyer, A. Papazoglou, A. Klein and A. Fahrner. We thank W. Wünsch for his help digitizing the videos. We also thank J. Loring for technical help with Supplementary Video 6 and A. Eroshkin for discussion on the mathematical model in the Supplementary Note. This work was supported by the Deutsche Forschungsgemeinschaft (Si874/2-1), the US National Institutes of Health, the American Parkinson's Disease Association, Children's Neurobiological Solutions, the March of Dimes, Margot Anderson Brain Restoration Foundation and Project ALS.
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Supplementary information
Supplementary Fig. 1
Beating cellular specializations of neurosphere cells. (PDF 102 kb)
Supplementary Note
Mathematical analysis of proliferative cells in secondary neurospheres. (PDF 131 kb)
Supplementary Video 1
Representative time-lapse video microscopy demonstrating frequent, rapid, and multiple “coalescence” of secondary neurospheres. Note that neurospheres are capable of active locomotion, apparently propelled by tiny beating cellular processes. The movie shows a ∼2.5 hr segment of the 10-hr time-lapse recording excerpted as well in Fig. 2. The video recorder captured images every 3.5 seconds. This neurosphere culture was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of 50 cells/μl and 104 cells/cm2 (standard “medium density”), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO2 at the time of this videomicrograph. For illustrative purposes, this movie presents an “overview” of a “medium” cell density culture in a 6-well plate. Numerous time-lapse video-microscopic recordings confirmed merging of spheres grown at even “clonal” cell density (Supplementary Video 2 online). The beating cellular processes are better appreciated at higher power in Supplementary Video 3 online. (MOV 1971 kb)
Supplementary Video 2
Time-lapse video-microscopy demonstrating, at higher magnification, neurosphere locomotion and “coalescence” despite plating at “clonal” density. Cellular processes (see also Supplemental Fig. 1 and Supplemental Video 3) propel neurospheres over considerable distances increasing the probability of coalescence events in even low density cultures. The movie shows a time-lapse recording of ∼2 hr. This neurosphere culture (24-well plate) was generated from the dissociated forebrain of a newborn mouse, initially seeded at a density of only 5 cells/μl and 103 cells/cm2 (“clonal” density), and cultured for 5 days in a humidified atmosphere at 37°C and 5% CO2 at the time of this videomicrograph. (MOV 676 kb)
Supplementary Video 3
Time-lapse video microscopy of human fetal cortex-derived neurospheres at higher magnification. Note the tiny beating cellular processes covering the surface of two merging human neurospheres. These beating structures were abundant on human and rodent neurospheres irrespective of their origin (e.g. cortex, striatum, spinal cord) and seem to propel them in suspension culture. (MOV 9235 kb)
Supplementary Video 4
Time-lapse video microscopy of human neurospheres grown in 96-well plates. Neurospheres are motile and merge in 96-well plates similar to the findings obtained in 6-well (Video 1) and 24-well (Video 2) plates. In this experiment with 96-well plates, the initial plating density was 10 cells/μl or 2000 cells/well (personal communication, B. Reynolds), but nevertheless still generated polyclonal spheres. Note that movie speed here is 5 times faster than in the other Videos. (MOV 6887 kb)
Supplementary Video 5
Time-lapse video microscopy of mouse cell culture showing neurosphere fusion and interaction of motile spheres with single cells attached to uncoated plastic dish. This example shows, besides the merging of two neurospheres, that single cells can attach to or detach from a motile neurosphere compromising their clonal boundaries even at “clonal” plating density (5 cells/μl). (MOV 2174 kb)
Supplementary Video 6
Time-lapse video microscopy of neurosphere-like cells that have become adherent. Note that adherent spheres, too, migrate and coalesce. Only if this concentrated culture dish had begun as a single isolated cell in a single isolated miniwell would an assumption of clonal boundaries and identity be justified and legitimate. In this experiment mouse forebrain stem cells (20 cells/μl) were plated into 24-well plates with glass coverslips coated with poly-L-lysine and laminin. The videomicrograph was taken on day 7. (MOV 5136 kb)
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Singec, I., Knoth, R., Meyer, R. et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 3, 801–806 (2006). https://doi.org/10.1038/nmeth926
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DOI: https://doi.org/10.1038/nmeth926
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