Epidermal structure created by canine hair follicle keratinocytes enriched with bulge cells in a three-dimensional skin equivalent model in vitro: Implications for regenerative therapy of canine epidermis

Background - Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. Hypothesis/Objectives - This study was designed to determine the ability of canine hair follicle bulge cell-enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. Animals - Four healthy beagle dogs from a research colony. Methods - Bulge cell-enriched keratinocytes showing keratin15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT-PCR analyses after 10-14days of culture at the air-liquid interface. Results - The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin14 and desmoglein3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin10 and desmoglein1 occurred in more superficial layers. Claudin1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. Conclusions and clinical importance - A bulge stem cell-enriched population from canine hair follicles formed interfollicular epidermis within 2weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.