Ability of normal human keratinocytes that grow in culture in serum-free medium to be derived from suprabasal cells.

The current studies were performed to determine from which regions of the skin keratinocytes that grow in vitro are derived. Normal human foreskin specimens were first separated by differential trypsinization into two suprabasal fractions and one basal fraction. Utilizing complete MCDB 153 basal nutrient culture medium containing epidermal growth factor and insulin, we then evaluated the clonogenic potential of cells in these three fractions. Suprabasal cell fractions demonstrated a colony-forming efficiency as great as or greater than that of the basal cell fraction, and 10%-15% of the keratinocytes that grew in primary and secondary cultures expressed involucrin, a suprabasal keratinocyte differentiation marker. Of such involucrin-containing keratinocytes, 80% also possessed the potential to undergo DNA synthesis, as determined by autoradiography following a 48-hour incubation with [3H]thymidine. These observations support the conclusion that the human keratinocytes that grow in vitro in serum-free medium can be derived from suprabasal cells and, therefore, that a state of nonterminal keratinocyte differentiation exists.