Homeostasis of the epidermis and skin appendages is maintained by tissue-specific stem cells. In mice and humans, two populations of epithelial stem cells have been identified: one in the basal layer of the interfollicular epidermis and another in the bulge area of hair follicles. However, our understanding of canine epithelial stem cells is extremely limited. In this study, in vitro colony-forming assays were performed to locate highly proliferative keratinocytes in canine skin. Their phenotypic resemblance to epithelial stem cells in other species was also assessed. When equal numbers of epidermal or hair-follicle keratinocytes were cultured, colonies derived from follicular keratinocytes were significantly larger both in total numbers and size, than those derived from epidermal keratinocytes. In addition, immunoreactivity for CD34, a putative bulge stem-cell marker in the mouse, was predominantly detected in follicular keratinocytes. Thus in dogs, follicular keratinocytes were distinct from epidermal keratinocytes in proliferative capacity and CD34 expression. Using microdissection, highly proliferative keratinocytes were located within the middle portion of hair follicles, including the bulge area. Immunohistochemical study revealed that keratin 15, an established marker of bulge stem cells in mice and humans, was also predominantly expressed in the canine bulge area. Flow cytometry analysis revealed high numbers of keratin-15-positive cells in the highly proliferative keratinocyte compartment. Of note, keratin 15high cells possessed the phenotypic characteristics of putative stem cells. This study represents the first in vitro identification and isolation of highly proliferative canine keratinocytes, which represent candidate epithelial stem cells.
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