Fig. 1. Characterization of the Adh⁺⁺⁺EGF-R^-/+ epidermal stem cell subpopulation. (A) Basal keratinocytes were separated on the basis of their adhesion properties. The cell population designated as Adh^-/+ is composed of keratinocytes with low adhesion capacity, and the Adh⁺⁺⁺ population is composed of keratinocytes with high adhesion capacity. Cells of each population were studied for their long-term expansion potential. Expansion curves are expressed as a cumulated cell output. Data represent means±s.d. of four replicate cultures from one typical experiment. (B) Cells of the Adh⁺⁺⁺ population were labeled to analyze their level of EGF-R cell-surface expression by flow cytometry. Sorting gates were defined to isolate four subpopulations: the Adh⁺⁺⁺EGF-R^-/+ subpopulation contains the 20% of the Adh⁺⁺⁺ keratinocytes presenting the lowest level of EGF-R expression; the Adh⁺⁺⁺EGF-R⁺⁺⁺⁺ subpopulation contains the 20% of the Adh⁺⁺⁺ keratinocytes presenting the highest level of EGF-R expression; the Adh⁺⁺⁺EGF-R⁺⁺ and Adh⁺⁺⁺EGF-R⁺⁺⁺ subpopulations each contained the 20% of the Adh⁺⁺⁺ keratinocytes presenting intermediate levels of EGF-R expression. (C) The long-term proliferative potential of Adh⁺⁺⁺EGF-R^-/+, Adh⁺⁺⁺EGFR⁺⁺, Adh⁺⁺⁺EGF-R⁺⁺⁺ and Adh⁺⁺⁺EGF-R⁺⁺⁺⁺ keratinocytes were compared. Data represent means±s.d. of four replicate cultures from one typical experiment. (D) Capacity of cell subpopulations, sorted according to the level of cell-surface EGF-R expression, to generate a reconstructed epidermis. Selected keratinocytes of the Adh⁺⁺⁺EGF-R^-/+ and Adh⁺⁺⁺EGF-R⁺⁺⁺⁺ subpopulations were expanded in defined culture conditions, and then seeded on to a dermal substrate at an early passage (p4) and a late passage (p7) to evaluate their capacity to produce a pluristratified epidermis. Histological preparations shown are from one typical experiment.