Mol. Hum. Reprod. Advance Access originally published online on November 7, 2006
Molecular Human Reproduction 2007 13(1):21-32; doi:10.1093/molehr/gal091
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Epithelial–mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions
1Research Centre Reproduction and Genetics, University Hospital and Medical School of the Vrije Universiteit Brussel (VUB, Free University of Brussels), 2Experimental Pathology Department, Medical School of the VUB, 3Laboratory of Developmental and Tumour Biology, University of Liège, 4Centre for Medical Genetics, University Hospital of the VUB and 5Centre for Reproductive Medicine, University Hospital of the VUB, Brussels, Belgium
6 To whom correspondence should be addressed at: Research Centre Reproduction and Genetics, University Hospital and Medical School of the VUB, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: urielle.ullmann{at}az.vub.ac.be
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Abstract |
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Feeder-free human embryonic stem cell (hESC) culture is associated with the presence of mesenchymal-like cells appearing at the periphery of the colonies. The aim of this study was to identify this early differentiation process. Long-term feeder-free hESC cultures using matrigel and conditioned medium from mouse and from human origin revealed that the appearance of mesenchymal-like cells was similar regardless of the conditioned medium used. Standard characterization confirmed the preservation of hESC properties, but the feeder-free cultures could not be maintained longer than 37 passages. The early differentiation process was characterized in the short term after switching hESCs cultured on feeders to feeder-free conditions. Transmission electron microscopy showed an epithelium-like structure inside the hESC colonies, whereas the peripheral cells revealed the acquisition of a rather mesenchymal-like phenotype. Immunochemistry analysis showed that cells at the periphery of the colonies had a negative E-cadherin expression and a positive Vimentin expression, suggesting an epithelial–mesenchymal transition (EMT). Nuclear staining of ß-catenin, positive N-cadherin and negative Connexin 43 expression were also found in the mesenchymal-like cell population. After RT–PCR analysis, Slug and Snail, both EMT-related transcription factors, were detected as up-regulated in the mesenchymal-like cell population. Taken together, our data suggest that culturing hESCs in feeder-free conditions enhances an early differentiation process identified as an EMT.
Key words: human embryonic stem cells/feeder-free culture/matrigel/differentiation/epithelial–mesenchymal transition
This study was presented orally at the 22nd Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE) in Prague (18–21 June 2006) (abstract O-236).
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