Journal
STEM CELLS
Volume 26, Issue 11, Pages 2938-2944Publisher
WILEY
DOI: 10.1634/stemcells.2008-0558
Keywords
Stem cell; Clonality; Tissue culture; Fluorescent mice
Categories
Funding
- Canadian Institutes of Health Research
- Natural Sciences and Engineering Research Council of Canada
- Canadian Stem Cell Network
- Ontario Graduate
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Recent reports have challenged the clonality of the neurosphere assay in assessing neural stem cell (NSC) numbers quantitatively. We tested the clonality of the neurosphere assay by culturing mixtures of differently labeled neural cells, watching single neural cells proliferate using video microscopy, and encapsulating single NSCs and their progeny. The neurosphere assay gave rise to clonal colonies when using primary cells plated at 10 cells/mu l or less; however, when using passaged NSCs, the spheres were clonal only if plated at 1 cell/mu l. Most important, moving the plates during the growth phase (to look at cultures microscopically) greatly increased the incidence of nonclonal colonies. To ensure clonal sphere formation and investigate nonautonomous effects on clonal sphere formation frequencies, single NSCs were encapsulated in agarose and proliferated as clonal free-floating spheres. We demonstrate that clonal neurospheres can be grown by avoiding movement-induced aggregation, by single-cell tracking, and by encapsulation of single cells. STEM CELLS 2008;26:2938-2944
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