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This Article Full Text Full Text (PDF) All Versions of this Article: 2008-0365v1 26/11/2777    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Google Scholar Articles by Van Hoof, D. Articles by Mummery, C. L. PubMed PubMed Citation Articles by Van Hoof, D. Articles by Mummery, C. L.

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Feeder-Free Monolayer Cultures of Human Embryonic Stem Cells Express an Epithelial Plasma Membrane Protein Profile

^aHubrecht Institute, Department of Developmental Biology and Stem Cell Research, Utrecht, The Netherlands;
^bBiomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands;
^cLeiden University Medical Center, Department of Anatomy and Embryology, Leiden, The Netherlands;
^dInteruniversity Cardiology Institute of The Netherlands and Heart Lung Institute, University Medical Centre Utrecht, Utrecht, The Netherlands

Key Words. Embryonic stem cell • Monolayer • Feeder-free • Epithelial • Mesenchymal • Cell-cell adhesion complex

Correspondence: Prof. Dr. Christine L. Mummery, Department of Anatomy and Embryology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Telephone: 31-71-526-9307; Fax: 31-71-526-8289; e-mail: c.l.mummery{at}lumc.nl

Received April 11, 2008; accepted for publication August 8, 2008.
First published online in STEM CELLS EXPRESS   August 14, 2008.

Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.