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

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

PROVIDER: PRJNA704653 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13777533.fastq.gz	Fastqsanger.gz
	SRR13777534.fastq.gz	Fastqsanger.gz
	SRR13777535.fastq.gz	Fastqsanger.gz
	SRR13777536.fastq.gz	Fastqsanger.gz
	SRR13777537.fastq.gz	Fastqsanger.gz

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Long-term expansion and differentiation of adult murine epidermal stem cells in three-dimensional organoid cultures

Project description: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.

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

Project description: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.

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