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

Culture of keratinocytes in conventional medium without a mesenchyme-derived 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 Green's medium, on plastic without a feeder layer, in order to investigate the degree of differentiation that they achieve in these conditions. Intermediate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemistry and electron microscopy. Before reaching confluence, keratinocytes expressed keratin molecules, as well as vimentin, and formed tonofibrils and desmosomes. The expression of these markers was progressively reduced until confluence and was totally lost thereafter, while cultures could be propagated for at least six passages. On the contrary, reseeding on a feeder layer after 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.