Enrichment for murine keratinocyte stem cells based on cell surface phenotype

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 [(3)H]thymidine ([(3)H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [(3)H]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 alpha(6) integrin and low to undetectable expression of the transferrin receptor (CD71) termed alpha(6)(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor ( approximately 8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing approximately 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 alpha(6)(bri)CD71(bri), representing the majority ( approximately 60%) of basal keratinocytes that are actively cycling, and importantly contain approximately 70% of [(3)H]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.

Figure 1

mAb 10G7 recognizes the human transferrin receptor (HTR) CD71. Flow cytometric analysis of CEF + HTR cells showing their reactivity with a known anti-CD71 antibody (B3/25.4) and mAb 10G7. mAb 1D4.5 is an isotype-matched negative control for mAb 10G7.

Figure 2

Two-color flow cytometric analysis of α₆ integrin and CD71 expression on primary murine dorsal keratinocytes. α₆ was detected with FITC (x axis, FL1) and CD71 with phycoerythrin (y axis, FL2). Three phenotypically distinct fractions of α₆ positive keratinocytes were consistently discernable (n = 25) as indicated: a, α₆^briCD71^bri cells making up the majority of basal keratinocytes; b, α₆^briCD71^dim cells representing a discrete but minor proportion of basal keratinocytes; and c, α₆^dim cells that appear as a less discrete population. A number of α₆ negative nonepithelial cells were also detected.

Figure 3

Cell cycle, cell size, and morphological characteristics of fractionated murine primary dorsal keratinocytes. (a) Cell cycle analysis from two independent sorting experiments demonstrated that the α₆^briCD71^bri fraction was enriched for actively cycling basal keratinocytes, with the fewest number of cells in G₀/G₁; conversely, the α₆^briCD71^dim fraction was the most quiescent subpopulation with the least number of cells in S and G₂/M, but the highest frequency of cells in G₀/G₁. The α₆^dim cells represented an intermediate population that was not significantly different from the α₆^briCD71^dim fraction (P = 0.126). (b and c) A comparison of the forward- and side-scatter dot plots of these two populations of basal keratinocytes shows that the quiescent α₆^briCD71^dim cells (b) are smaller in size as compared with the actively cycling α₆^briCD71^bri cells (c). (d and e) Giemsa-stained cytospin preparations of these fractions confirm these size differences as well as demonstrating that the α₆^briCD71^dim population has a high nuclear:cytoplasmic ratio (d) as compared with the α₆^briCD71^bri population (e). These properties indicate enrichment for putative stem cells in the α₆^briCD71^dim fraction.

Figure 4

Enrichment for [³H]Tdr LRCs in the α₆^briCD71^dim and PLCs in the α₆^briCD71^bri compartments. The results displayed represent the percentage of LRCs/PLCs found in the three phenotypically distinct keratinocyte fractions, expressed as a proportion of the total labeled cells displayed as mean ± SD.

Figure 5

The bulge region of the hair follicle is CD71^dim. (a) Immunofluorescence micrographs of CD71 staining (green) in dorsal skin illustrating several early anagen hair follicles (arrowheads), with bright staining for CD71 at the base of the follicle. Note the longitudinal section through a mid-anagen hair follicle showing relatively low to negative CD71 expression in the bulge region (block arrows), directly below the sebaceous glands (arrow). (b and c) Dual immunofluorescence for CD71 (green) and nuclei stained with propidium iodide (red) illustrating the presence of CD71^dim cells in the bulge region (b) and CD71^bri cells in the bulb region (c). Dermal papilla is indicated by the star.