Markers of epidermal differentiation expressed by rat keratinocytes cultured by a modified feeder layer technique

A broad range of analytical methods has been used to investigate the expression of key differentiation markers in keratinocytes cultured by a modified feeder layer technique. Cultures were stratified and showed many of the features characteristic of epidermal differentiation in vivo including tonofilaments, desmosomes, loss of organelles and thickening of the plasma membrane to form the cornified envelope. Profilaggrin synthesis was detected by 32P-incorporation and the presence of filaggrin suggested that it was broken down by the normal route. Staining with the lectin from Ulex europeus revealed the presence of a fucose-containing cell-surface glycoprotein. Keratin synthesis was shown by 3H-leucine incorporation and keratins were analysed by two-dimensional gel electrophoresis in comparison with those from different levels of the epidermis. Quantitative and qualitative differences were found between in vivo and in vitro epidermal differentiation. In particular, cornified envelope numbers were low, in keeping with the observation by electron microscopy of only one layer of cells with this structure. The absence of a true stratum corneum in vitro was also indicated by the virtual absence of histidase activity and stratum corneum keratins. The keratin species present in vitro most closely resembled those of the basal cells of the epidermis, although even in this case differences were observed. The evidence as a whole is consistent with the belief that epidermal cells do synthesise in vitro many of the important proteins involved in differentiation, but that they nevertheless do not develop a true keratinised stratum corneum.