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Original Article |
1 Cellartis AB, Göteborg, Sweden
2 TATAA Biocenter, Göteborg, Sweden
3 Buck Institute for Age Research, Novato, California
4 National Institute of Aging, Baltimore, Maryland
5 Lund University, Lund, Sweden
* To whom correspondence should be addressed. E-mail: peter.sartipy{at}cellartis.com.
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Abstract |
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There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESC). Using light microscopy and immunohistochemistry, we observed that morphological changes of differentiating hESC 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 hESC we developed a robust and sensitive multimarker quantitative real time PCR (QPCR) method. In order to maximize the sensitivity of the method we measured the expression of up- and down regulated genes before and after differentiation of the hESC. Out of the twelve 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 (TDGF1), and 
-fetoprotein (AFP). 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 hESC in, for example, pharmacological and cytotoxicity screening.
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