Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions.
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 beta-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.
Ullmann
U
U
Research Centre Reproduction and Genetics, University Hospital and Medical School of the Vrije Universiteit Brussel, VUB, Free University of Brussels, Brussels, Belgium. urielle.ullmann@az.vub.ac.be
In't Veld
P
P
Gilles
C
C
Sermon
K
K
De Rycke
M
M
Van de Velde
H
H
Van Steirteghem
A
A
Liebaers
I
I
D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D015820:Cadherins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C000604407:SNAI1 protein, human; C000604449:Snai2 protein, mouse; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D014746:Vimentin