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ORIGINAL ARTICLE-CHARACTERIZATION SERIES

Monitoring Differentiation of Human Embryonic Stem Cells Using Real-Time PCR

^a Cellartis AB, Göteborg, Sweden;
^b TATAA Biocenter, Lundberg Laboratory, Göteborg, Sweden;
^c Cellular Neurobiology Branch, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland, USA;
^d Laboratory of Neurosciences, National Institute of Aging, Department of Health and Human Services, Baltimore, Maryland, USA;
^e Section of Endocrinology, Lund University, Lund, Sweden

Key Words. Human embryonic stem cell • Differentiation • Real-time polymerase chain reaction • Gene expression

Correspondence: Peter Sartipy, Ph.D., Cellartis AB, Arvid Wallgrens Backe 20, 413 46 Göteborg, Sweden. Telephone: 46-(0)31-7580930; Fax: 46-(0)31-7580910; e-mail: peter.sartipy{at}cellartis.com

There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs). Using light microscopy and immunohistochemistry, we observed that morphological changes of differentiating hESCs precede any major alterations in the expression of several commonly used hESC markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and Nanog). In an attempt to quantify the changes during stochastic differentiation of hESCs, we developed a robust and sensitive multi-marker quantitative real-time polymerase chain reaction (QPCR) method. To maximize the sensitivity of the method, we measured the expression of up- and downregulated genes before and after differentiation of the hESCs. Out of the 12 genes assayed, we found it clearly sufficient to determine the relative differentiation state of the cells by calculating a collective expression index based on the mRNA levels of Oct-4, Nanog, Cripto, and -fetoprotein. We evaluated the method using different hESC lines maintained in either feeder-dependent or feeder-free culture conditions. The QPCR method is very flexible, and by appropriately selecting reporter genes, the method can be designed for various applications. The combination of QPCR with hESC-based technologies opens novel avenues for high-throughput analysis of hESCs in, for example, pharmacological and cytotoxicity screening.

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