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

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 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 stressinduced response, unrelated to differentiation. STEM

Epithelial–mesenchymal transition in rhesus monkey embryonic stem cell colonies: the role of culturing conditions

“…This is not surprising since ESCs form monolayers on laminin instead of multilayered colonies, and they take up a phenotype somewhat intermediary between epithelial and mesenchymal, which is consistent with observations made by other authors using various laminin isotypes (Domogatskaya et al 2008;Miyazaki et al 2008) or Matrigel (Ullmann et al 2007(Ullmann et al , 2008Van Hoof et al 2008). In the latter case, a diffuse EMT without localized ICs was reported to occur at the margins of the colonies.…”

Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation

“…27,28 When used for coculture, 3D approaches have varied, including the formation of spheroids with multiple cell types, 29 encapsulated protein gels seeded with multiple cell types, 30 or hybrid methods of 3D cultures and monolayers. 31 In general, these methods have been successful in creating improved in vivo-like conditions, yet these methods often lack the spatial control necessary to organize cells into complex organotypic models that recapitulate tissue environments.…”

Protein Kinase C-Induced Early Growth Response Protein-1 Binding to SNAIL Promoter in Epithelial–Mesenchymal Transition of Human Embryonic Stem Cells

“…Several transcription factors have been implicated in this repression, including SNAIL, SLUG, and TWIST1 [32][33][34][35][36][37]. Actually, in hPS cell culture, spontaneous differentiation is sometimes observed with the expression of SNAIL and VIMENTIN at the peripheral of the hPS cell colonies in conventional culture conditions, indicating that EMT occurs in hPS cell culture [38][39][40][41].…”