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
Authors
Tetsuro Kobayashi, Kaoru Enomoto, Yu Hsuan Wang, Ji Seon Yoon, Ryoko Okamura, Kaori Ide, Manabu Ohyama, Toshio Nishiyama, Toshiroh Iwasaki and Koji Nishifuji
Institution
Keio University
Country
Japan
Year
2013
Journal
Vet Dermatol
Abstract
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 cellenriched
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 keratin 15 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 10–14 days 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 keratin 14 and desmoglein 3 were detected in
the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in
more superficial layers. Claudin 1 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 2 weeks in vitro, and thus represents a promising model for regenerative therapy
of canine skin.