Long-Term Expansion of Mouse Primary Epidermal Keratinocytes Using a TGF-β Signaling Inhibitor

Mouse models have been used to study the physiology and pathogenesis of the skin. However, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce a newly developed protocol that enables long-term expansion of p63⁺ mouse epidermal keratinocytes in low Ca²⁺ media without the need of progenitor cell-purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-β signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63⁺ epidermal progenitor cells, while preserving their ability to differentiate. Suppression of TGF-β signaling also permits p63⁺ epidermal keratinocytes to form macroscopically large clones in 3 T3-J2 feeder cell co-culture. This simple and efficient approach will facilitate the use of mouse models by providing p63⁺ primary epidermal keratinocytes in quantity.