Long-term expansion and differentiation of adult murine epidermal stem cells in three-dimensional organoid cultures

Mammalian epidermal stem cells maintain homeostasis of skin epidermis and contribute to its regeneration throughout adult life. While two-dimensional mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cyclic AMP, FGF and R-spondin signaling with inhibition of BMP signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 months, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.