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

¹ Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
² Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; Leiden University Medical Center, Department of Anatomy and Embryology, Einthovenweg 20, 2333 ZC Leiden, The Netherlands
³ Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
⁴ Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; Leiden University Medical Center, Department of Anatomy and Embryology, Einthovenweg 20, 2333 ZC Leiden, The Netherlands; Interuniversity Cardiology Institute of The Netherlands, and Heart Lung Institute, University Medical Centre Utrecht, 3584 CX Utrecht, The Netherlands

^* To whom correspondence should be addressed. E-mail: c.l.mummery{at}lumc.nl.

	Abstract

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 multi-layered 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 show that hESCs under these conditions primarily express proteins belonging to epitheliumrelated 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 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 (EMT), indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when 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.