A Simplified Protocol for Isolation and Culture of Keratinocyte Stem Cells from Neonatal Mice.

Isolation of primary keratinocyte stem cells (KSCs) from neonatal mouse epidermis is essential for studying skin physiology and related disorders. Traditional methods often struggle to balance keratinocyte proliferation and differentiation, and although recent advancements using low-calcium culture conditions have improved these techniques, protocols remain scattered. This study presents a streamlined approach to expand mouse KSCs in low-calcium medium (<0.07 mM calcium), promoting proliferation while preserving stem cell properties and enabling controlled differentiation, all without the need for feeder cells. Conditioned medium derived from primary dermal fibroblasts (DFs), isolated from the same neonatal mice used for KSC isolation, was developed to enhance KSC proliferation and maintain stem cell characteristics. This fibroblast-conditioned medium significantly boosted KSC expansion and supported both proliferation and differentiation. A two-step purification process, based on rapid stem cell attachment to a composite matrix, ensured high cell purity and eliminated interference from other epidermal cell populations, making the approach reliable and effective. By eliminating feeder layers and employing fibroblast-conditioned medium, this optimized protocol facilitates the accessibility of primary KSCs for research, supporting investigations into skin disorders and signaling pathways, and advancing progress in skin biology.