Targeted inhibition of wound-induced PAI-1 expression alters migration and differentiation in human epidermal keratinocytes.

In the adult epidermis, keratinocytes do not normally express the type-1 inhibitor of plasminogen activator (PAI-1). Basal epithelial cell-specific PAI-1 synthesis, however, accompanies epidermal wound repair in vivo in which PAI-1 transcripts and immunoreactive protein are confined to epithelial cells in the migrating tongue and the hyperproliferative zone. A model system using human keratinocytes (HaCaT cells) was developed to assess functional relationships between epithelial growth state transitions and PAI-1 expression.

PAI-1 gene expression is growth state-regulated in cultured human epidermal keratinocytes during progression to confluence and postwounding.

Growth of human keratinocytes (NHKs) in submerged cultures approximates a "wound" response generally considered equivalent to regenerative maturation. Within this context, PAI-1 expression in cultured NHKs appears to be growth state-regulated and is associated with specific NHK subpopulations undergoing migration and proliferation in response to wounding; NHKs transit through specific phases during growth to confluence. Basal layer keratinocytes comprise several classes of nucleated epidermal cells (designated "A," "B," and "C") which are distinguishable on the basis of RNA content, population generation time, and expression of basal cell marker proteins.

Growth of melanocytes in human epidermal cell cultures.

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin.

Sodium-N-butyrate induces cytoskeletal rearrangements and formation of cornified envelopes in cultured adult human keratinocytes.

The technique developed in our laboratory allows us to culture multilayered, stratified sheets of human keratinocytes, which can be used to cover the burn wounds of patients. Organization of cells in these cultures resembles stratum germinativum and stratum spinosum but there are only a few fully keratinized cells and the stratum corneum is not developed. Since the fully differentiated sheets may offer additional advantages as epidermal transplants, attempts were made to enhance the degree of differentiation in vitro.

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics.