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Monitoring differentiation of human embryonic stem cells using real-time PCR

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Author

  • K Noaksson
  • N Zoric
  • XM Zeng
  • MS Rao
  • J Hyllner
  • Henrik Semb
  • M Kubista
  • P Sartipy

Summary, in English

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 multimarker 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 (x-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. STEM CELLS 2005;23:1460-1467.

Publishing year

2005

Language

English

Pages

1460-1467

Publication/Series

Stem Cells

Volume

23

Issue

10

Document type

Journal article

Publisher

AlphaMed Press

Topic

  • Cell and Molecular Biology

Keywords

  • real-time polymerase chain
  • human embryonic stem cell
  • differentiation
  • gene expression
  • reaction

Status

Published

ISBN/ISSN/Other

  • ISSN: 1549-4918