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

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 epi-dermis 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 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.

Abnormal keratinocyte differentiation in the nasal planum of Labrador Retrievers with hereditary nasal parakeratosis (HNPK)

“…The epidermis of the canine nasal planum is on average more than twice the thickness of haired skin in healthy dogs and displays an orthokeratotic SC but focal parakeratosis can occur [14]. One study showed that the healthy canine nasal planum displays a similar pattern of the differentiation markers K14 and K10, desmogleins 1 and 3, and filaggrin, compared to a canine in vitro three-dimensional skin equivalent [15]. Another study reported on comparable mRNA and protein expression of K5 and K10, filaggrin and involucrin in various haired body regions of dogs [16].…”

Basic principles of hair follicle structure, morphogenesis, and regeneration

“…94,99 This protrusion is not seen in domestic animals nor in humans, but the term bulge has been used in several publications and refers to the isthmus region. 46,47 In dogs, different SC populations have been identified along the entire isthmus. 25 Like in the dog, the SC bearing regions in the human HF are not yet well defined.…”

Ovine Hair Follicle Stem Cells Derived from Single Vibrissae Reconstitute Haired Skin

“…Kobayashi et al dissected and cultured the bulge area of canine hair follicles in vitro and obtained highly proliferative keratinocytes, which shared the same marker expression with mouse and human HFSCs [ 17 , 18 ]. In vitro , these canine HFSCs formed an IFE structure comprising cell layers that expressed markers similar to the normal epidermis [ 19 ].…”