We describe an improved culturing protocol to direct embryonic stem (ES) cell differentiation along the epidermal lineage in vitro. Pluripotent ES cells co-cultured with mitomycin C-treated mature epidermal cells at low density resulted in colonies that undergo differentiation to keratin 14 (K14)-positive cells within 3 d in culture. This new protocol suggests that the commitment and differentiation of undifferentiated ES cells can be efficiently directed by mature epidermal cell-derived factors. In addition, it suggests that this condition may be responsible for the maintenance and expansion of putative epidermal stem cells in vitro. Our advanced culturing regimen allows for the analysis of colony formation from a single cell to determine the factors controlling the differentiation capacities of very early progenitor cells in culture.
