Isolation and long-term expansion of murine epidermal stem-like cells

Epidermis is the most outer layer of the skin and serves as a physical barrier protecting the internal tissues from mechanical and environmental insults. Its constant renewing and regeneration capacity are sustained by the basal keratinocytes, which, through proliferation and differentiation, supply diverse cell types for homeostasis maintenance and injury repair. A major challenge in epidermal research is the difficulty to culture murine keratinocytes. Here we tested murine keratinocyte culture conditions and developed cell lines using low-Ca2+ (0.06 mM) keratinocyte serum-free medium (KSFM-Ca2+) without feeder cells. Cells derived in this condition could be subcultured for >70 passages. These cells displayed basal epithelial cell morphology and expressed keratin (KRT) 14, but lacked the epithelial-characteristic intercellular junctions. After adaptation to the Defined-KSFM (DKSFM) media containing 0.15 mM Ca2+, these cells formed tight- and adherens-junctions and exhibited increased Krt1/10 expression while retaining subculture capacity. Global gene expression studies showed that cells derived in KSFM-Ca2+ media had enriched expression of stem/proliferation markers and that cells adapted in DKSFM media had gene expression signatures of epithelial progenitors. Correspondingly, the KSFM-Ca2+-derived cells had a remarkable capacity of clonal growth, whereas DKSFM-adapted cells could differentiate to suprabasal subtypes in 3D cultures. The generation of stem-like murine keratinocyte lines and the conversion of these cells to epithelial progenitors capable of terminal differentiation provide useful in vitro tools for skin research. Gene exepression prolfiles of two newly established murine keratinocyte lines