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

Noaksson, K; Zoric, N; Zeng, XM; Rao, MS; Hyllner, J; Semb, Henrik LU ; Kubista, M and Sartipy, P (2005) In Stem Cells 23(10). p.1460-1467
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
real-time polymerase chain, human embryonic stem cell, differentiation, gene expression, reaction
in
Stem Cells
volume
23
issue
10
pages
1460 - 1467
publisher
AlphaMed Press
external identifiers
  • wos:000233708700005
  • scopus:28444485641
ISSN
1549-4918
DOI
10.1634/stemcells.2005-0093
language
English
LU publication?
yes
id
707dff2e-2978-4204-b798-40bbb621e236 (old id 211310)
date added to LUP
2007-08-20 09:04:21
date last changed
2017-01-01 07:17:09
@article{707dff2e-2978-4204-b798-40bbb621e236,
  abstract     = {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.},
  author       = {Noaksson, K and Zoric, N and Zeng, XM and Rao, MS and Hyllner, J and Semb, Henrik and Kubista, M and Sartipy, P},
  issn         = {1549-4918},
  keyword      = {real-time polymerase chain,human embryonic stem cell,differentiation,gene expression,reaction},
  language     = {eng},
  number       = {10},
  pages        = {1460--1467},
  publisher    = {AlphaMed Press},
  series       = {Stem Cells},
  title        = {Monitoring differentiation of human embryonic stem cells using real-time PCR},
  url          = {http://dx.doi.org/10.1634/stemcells.2005-0093},
  volume       = {23},
  year         = {2005},
}