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TISSUE-SPECIFIC STEM CELLS

Don't Look: Growing Clonal Versus Nonclonal Neural Stem Cell Colonies

Departments of ^aMolecular Genetics,
^bSurgery, Division of Anatomy, and
^dChemical Engineering and Applied Chemistry and
^cInstitute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

Key Words. Stem cell • Clonality • Tissue culture • Fluorescent mice

Correspondence: Correspondence: Derek van der Kooy, Ph.D., University of Toronto, Dept Molecular Genetics, TD-CCBR, Room 1102, 160 College Street, Toronto, Ontario M5S 3E1, Canada. Telephone: 416-978-1960; Fax: 416-978-2666; e-mail: Derek.van.der.kooy{at}utoronto.ca

Received on June 9, 2008; accepted for publication on August 11, 2008.

First published online in STEM CELLS EXPRESS  August 28, 2008.

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/µl or less; however, when using passaged NSCs, the spheres were clonal only if plated at 1 cell/µl. 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.

Disclosure of potential conflicts of interest is found at the end of this article.

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