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. Overall design: We establish an organoid culture system for long-term expansion of mouse epidermal stem cells. Using histological methods as well as low-coverage multiplexed RNA sequencing, we show that cultured organoids resembled interfollicular epidermis. We analyzed a total of 23 samples, including 6 controls that are isolated from the skin of mice. None-passaged as well as cultured organoids were compared with replicates. Differences growth factors and small molecules that allow expansion of organoids were compared with replicates.

23

Boonekamp KE, Kretzschmar K, Wiener DJ, Asra P, Derakhshan S, Puschhof J, López-Iglesias C, Peters PJ, Basak O, Clevers H