Abstract
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-Ca2+ (0.06 mM) keratinocyte serum-free medium (KSFM-Ca2+) 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 Ca2+, 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-Ca2+ media had enriched stem/proliferation markers and cells adapted in DKSFM media had epithelial progenitor signatures. Correspondingly, KSFM-Ca2+-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.
Highlights
Epidermis renews and regenerates continuously throughout the lifetime of an organism
Because calcium ion is crucial for keratinocyte proliferation and differentiation [16], we measured Ca2+ levels in three commercial media: (i), KSFM without Ca2+ (KSFM-Ca2+), (ii), Fig 1
We describe the establishment of murine epithelial cell lines under conditions free of serum and feeder cells using media containing minimal Ca2+ (KSFM-Ca2+)
Summary
Epidermis renews and regenerates continuously throughout the lifetime of an organism. The keratinocyte, located at the basal layer, is the primary cell type in the epidermis that divides and serves as a source for epidermal maintenance and regeneration through self-renew and periodical differentiation. The cells move outwards in a process known as basal-to-suprabasal transition to replace the cells at outer layer that are sloughed off into the environment. Differentiation is associated with up-regulation of signature genes, such as Krt, Krt, Involucrin and Loricrin. These gene products form the intermediate filament network that tightens cell junctions at the epidermal surface to enable protection against the hostile environment.
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