Human Epidermis Reconstructed by Culture: Is It “Normal”?

Epidermal differentiation: the bare essentials.

“…Tritiated thymidine labeling and examination of mitotic indices reveal a single layer of cuboidal-shaped basal cells, which express K5 and K14 (Kopan and Fuchs, 1989). As cells leave this layer, they synthesize spinous proteins, including K1 and involucrin (Asselineau et al, 1986;Kopan et al, 1987;see Fig. 1, B and C, respectively).…”

“…Thus, when seeded on a lawn of x-irradiated or mitomycin C-treated fibroblasts, and in medium containing the appropriate serum lot, cAMP-inducing agent, growth factors, and various additional nutrients, epidermal cells grow optimally for several hundred generations in culture. Despite the desirability of this system for generating rapidly growing keratinocytes, it is not optimal for differentiation, since only a few layers of cells form at confluence, and many of the biochemical changes characteristic of terminal differentiation, such as K1, K10 and filaggrin expression, do not occur (Fuchs and Green, 1980;Asselineau et al, 1986).…”

“…A clue for retaining these earlier stages of epidermal differentiation in culture came from studies by researchers culturing epidermal cells on floating lattices of collagen at the air-liquid interface (see Asselineau et al, 1986 for previous references). In initial attempts, basal cells were often flattened rather than cuboidal, and no visible granular layer was present.…”

“…Despite these shortcomings, four to five layers of spinous-like cells and four to five layers of comified cells were consistently obtained with floating cell cultures. From these studies, it was clear that when epidermal cells are exposed to the air and receive their nutrients via diffusion through an artificial dermis, their differentiation is significantly enhanced (Asselineau et al, 1986).…”

“…Indeed during mammalian development, expression of TGF-/32 mRNA (Pelton et al, 1989) and a related Vgr-1 mRNA Rheinwald (1980). Cells were transferred to a dish containing a lattice of collagen and fibroblasts, and at confluence, the epidermal cells and the lattice were raised to the air-liquid interface, and cultured for an additional three weeks (Asselineau et al, 1986;Kopan et al, 1987). The "tissue" was then fixed in Carnoy's solution, embedded in paraffin, and sectioned (5/~m).…”