Enrichment for murine keratinocyte stem cells based on cell surface phenotype

Hiroaki Tani, Rebecca J. Morris, Pritinder Kaur

Research output: Contribution to journalArticle

330 Scopus citations

Abstract

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor (≃8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≃70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6(bri)CD71(bri), representing the majority (≃60%) of basal keratinocytes that are actively cycling, and importantly contain ≃70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71(dim) cells in the hair follicle bulge region, a well documented location for KSCs.

Original languageEnglish (US)
Pages (from-to)10960-10965
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number20
DOIs
StatePublished - Sep 26 2000
Externally publishedYes

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