GSK-3-specific inhibitor-supplemented hESC medium prevents the epithelial-mesenchymal transition process and the up-regulation of matrix metalloproteinases in hESCs cultured in feeder-free conditions.
Feeder-free culture induces spontaneous differentiation of human embryonic stem cells (hESCs), identified as an epithelial to mesenchymal transition (EMT). The maintenance of pluripotency of hESCs in feeder-free cultures through the activation of the WNT pathway using a glycogen synthase kinase (GSK)-3-specific inhibitor (BIO) was reported. The aim of this study was to determine the effect of BIO on the EMT process. In contrast with those grown in feeder-free conditions with control medium, hESC colonies cultured with BIO-supplemented hESC medium did not show any fibroblast-like cells at the periphery. Transmission electron microscopy, relative quantitative real-time RT-PCR and immunostaining analyses showed the presence of epithelial features and a diminution of mesenchymal features in the BIO-treated hESCs such as a strong E-cadherin expression, the down-regulation of Vimentin, Snail and Slug expressions and a cytoplasmic beta-catenin expression. An up-regulation of matrix metalloproteinases (MMP) MMP-2, MMP-9, MT-1MMP (membrane-type 1 MMP) and EMMPRIN (extracellular MMP inducer) expression was also found associated with the EMT occurring in feeder-free hESCs cultures using mouse embryonic fibroblasts conditioned medium (MEF CM). The presence of BIO clearly down-regulated the expression of these MMPs. This study showed that BIO, a GSK-3-specific inhibitor, prevents the EMT process which is associated with the feeder-free hESC culture. Nevertheless, BIO was not sufficient to expand hESCs in a long-term culture system.
Ullmann
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U
Department of Embryology and Genetics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. urielle.ullmann@uzbrussel.be
Gilles
C
C
De Rycke
M
M
Van de Velde
H
H
Sermon
K
K
Liebaers
I
I
D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D010091:Oximes / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C483321:6-bromoindirubin-3'-oxime; D004791:Enzyme Inhibitors; D007211:Indoles; D010091:Oximes; D038362:Glycogen Synthase Kinase 3; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9