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

Epidermis is the most outer layer of the skin and a physical barrier protecting the internal tissues from mechanical and environmental insults. The basal keratinocytes, which, through proliferation and differentiation, supply diverse cell types for epidermal homeostasis and injury repair. Sustainable culture of murine keratinocyte, however, is a major obstacle. Here we developed murine keratinocyte lines using low-Ca(2+) (0.06 mM) keratinocyte serum-free medium (KSFM-Ca(2+)) without feeder cells. Cells derived in this condition could be subcultured for >70 passages. They displayed basal epithelial cell morphology and expressed keratin (Krt) 14, but lacked the epithelial-characteristic intercellular junctions. Moreover, these cells could be adapted to grow in the Defined-KSFM (DKSFM) media containing 0.15 mM Ca(2+), and the adapted cells established tight- and adherens-junctions and exhibited increased Krt1/10 expression while retained subculture capacity. Global gene expression studies showed cells derived in KSFM-Ca(2+) media had enriched stem/proliferation markers and cells adapted in DKSFM media had epithelial progenitor signatures. Correspondingly, KSFM-Ca(2+)-derived cells exhibited a remarkable capacity of clonal expansion, whereas DKSFM-adapted cells could differentiate to suprabasal epithelial cell types in 3-dimentional (3D) organoids. The generation of stem-like murine keratinocyte lines and the conversion of these cells to epithelial progenitors capable of terminal differentiation provide the critically needed resources for skin research.