human embryonic stem cells; feeder-free culture; matrigel; differentiation; epithelial–mesenchymal transition
Abstract :
[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Ullmann, U.
In'T Veld, P.
Gilles, Christine ; Université de Liège - ULiège > Département des sciences cliniques > Labo de biologie des tumeurs et du développement
Sermon, K.
De Rycke, M.
Van de Velde, H.
Van Steirteghem, A.
Liebears, I.
Language :
English
Title :
Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions.
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