Human keratinocytes cultured without a feeder layer undergo progressive loss of differentiation markers

Culture of keratinocytes in conventional medium without a mesenchyme-derive d feeder layer leads to poor growth and impaired differentiation; however, the exact pathway and degree of differentiation achieved in such conditions is unclear. We have cultured normal human keratinocytes in Rheinwald and G reen's medium, on plastic without a feeder layer, in order to investigate t he degree of differentiation that they achieve in these conditions. Interme diate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemist ry and electron microscopy. Before reaching confluence, keratinocytes expre ssed keratin molecules, as well as vimentin, and formed tonofibrils and des mosomes. The expression of these markers was progressively reduced until co nfluence and was totally lost thereafter, while cultures could be propagate d for at least six passages. On the contrary, reseeding on a feeder layer a fter the first passage led to rapid cell death. It could be concluded that signals from a feeder layer are relevant to support continuous synthesis of intermediate filaments proteins and formation of tonofibils and desmosomes , and that the derangement of the cytoskeleton in these conditions leads to altered, not simply defective, response to delayed stimulation by a feeder layer.