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This Article Free via Open Access: OA OA Full Text Full Text (PDF) Supplemental Data OA All Versions of this Article: 2007-0801v1 26/5/1109    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by O'Connor, M. D. Articles by Eaves, C. J. Search for Related Content PubMed PubMed Citation Articles by O'Connor, M. D. Articles by Eaves, C. J.

EMBRYONIC STEM CELLS

Alkaline Phosphatase-Positive Colony Formation Is a Sensitive, Specific, and Quantitative Indicator of Undifferentiated Human Embryonic Stem Cells

^aTerry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada;
^bSamuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

Key Words. Human embryonic stem cell • Pluripotent • Colony-forming cell • Differentiation • SSEA3 • OCT4

Correspondence: Michael O'Connor, Ph.D., Terry Fox Laboratory, 675 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. Telephone: 604-675-8000; Fax: 604-877-0712; e-mail: moconnor{at}bccrc.ca

Received September 20, 2007; accepted for publication February 6, 2008.
First published online in STEM CELLS EXPRESS   February 14, 2008.



Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP⁺) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of 30 AP⁺ cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3⁺ cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP⁺ clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.

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