Enrichment for murine keratinocyte stem cells based on cell surface phenotype

Area of Science:

• Dermatology
• Stem Cell Biology
• Cell Biology

Background:

• Epithelial stem cells are vital for tissue homeostasis, wound healing, and cancer.
• Distinguishing stem cells from transit amplifying (TA) cells is challenging due to a lack of specific markers.
• Cell kinetic analysis identifies murine keratinocyte stem cells (KSCs) as label-retaining cells (LRCs) and TA cells as pulse-labeled cells (PLCs).

Purpose of the Study:

• To physically isolate and characterize epithelial stem cells (KSCs) from their progeny (TA cells).
• To develop a method combining cell kinetic analysis and fluorescence-activated cell sorting for stem cell separation.
• To identify specific cell surface markers for distinguishing KSCs from TA cells.

Main Methods:

• In vivo cell kinetic analysis using [(3)H]thymidine ([(3)H]Tdr) labeling to identify label-retaining cells (LRCs) and pulse-labeled cells (PLCs).
• Fluorescence-activated cell sorting (FACS) to separate cell populations based on alpha(6) integrin and CD71 expression.
• Immunostaining of dorsal skin to localize identified cell populations.

Main Results:

• A subpopulation of alpha(6)integrin(high)CD71(low) (alpha(6)(bri)CD71(dim)) cells was identified, enriched for KSCs (approximately 8% of basal cells), characterized by quiescence and high nuclear:cytoplasmic ratio.
• This alpha(6)(bri)CD71(dim) population contained approximately 70% of label-retaining cells (LRCs).
• A distinct alpha(6)integrin(high)CD71(high) (alpha(6)(bri)CD71(bri)) subpopulation, representing the majority (approximately 60%) of basal keratinocytes, was enriched for actively cycling TA cells and contained approximately 70% of pulse-labeled cells (PLCs).
• CD71(dim) cells were found in the hair follicle bulge region, a known KSC niche.

Conclusions:

• Fluorescence-activated cell sorting, combined with cell kinetic analysis, successfully separates murine keratinocyte stem cells (KSCs) from transit amplifying (TA) cells.
• The alpha(6)(bri)CD71(dim) cell population represents quiescent KSCs, while the alpha(6)(bri)CD71(bri) population represents actively cycling TA cells.
• These distinct cell surface markers provide a means to isolate and study KSCs, advancing our understanding of epithelial stem cell biology, skin regeneration, and carcinogenesis.