Bovine coronary region keratinocyte colony formation is supported by epidermal-dermal interactions

Cross-section of the epidermis and dermal of the bovine coronary region (hematoxylin and eosin-stained; 40× magnification). A) Note the absence of inflammatory cell infiltrates, hemorrhage and lymphocytic infiltrates in the dermal (**) and overlying epidermal (*) regions. Immunohistochemical labeling was performed to determine the distribution of the keratinocyte basal cell marker, p63 in the epidermis (*) and dermis (**) of the bovine coronary region. Note that p63 distribution is greatest in basal layers at the epidermal-dermal junction in sections where the plane of the section is perpendicular (arrow) or parallel (arrow) to the long axis of the coronary region papilla; B) Picture representative of bovine coronary region epidermis and dermis.

Keratinocyte—fibroblast cocultures 7 d postisolation. Coronary region keratinocytes were overlaid onto 80 to 90% confluent monolayers of dermal fibroblast in Eagle's minimum essential medium supplemented with 10.0% fetal bovine serum. Keratinocytes appeared as islets of cobblestone-shaped cells surrounded by swirls of elongated, stellate-appearing fibroblast monolayers walling off a keratinocyte islet (panel A; 100× magnification). Islets of keratinocytes also formed stacks of cobblestone keratinocytes (panel B; 40× magnification). Pictures represent culture results from 1 of 40 cell preparations.

Cell identification with vimentin and cytokeratin immunofluorescent staining. Fibroblasts (panel A; 40× magnification) were identified by the cell marker vimentin using Cy3-conjugated murine anti-vimentin. Keratinocytes (panel C; 100× magnification) were identified by expression of keratin with a primary guinea pig anti-bovine fetal hoof keratin and a secondary fluorescein isothiocyanate-conjugated goat anti-guinea pig IgG. Fibroblasts (panel B; 100× magnification) demonstrated a diffuse and linear red fluorescence of the cytoplasm, whereas keratinocytes (panel D; 100× magnification) demonstrated a green fluorescence diffusely distributed throughout the cytoplasm. Pictures represent culture results from 1 of 40 cell preparations.

Coronary region keratinocyte colony appearance in keratinocyte—fibroblast coculture (40× magnification). Keratinocytes and fibroblasts were plated in coculture conditions at varying cellular inputs (digital pictures recorded clone growth for the plated concentration of 5.0×10³ keratinocytes/cm²) at 2, 6, and 10 d post-incubation. The number and size of colonies present showed a direct correlation to the increase in days, indicating that colony formation was a time-dependent process. At 2 d post-incubation (panel A), a confluent monolayer of fibroblasts (*) underlying small colonies of keratinocytes (arrow) was observed. At 6 d post-incubation (panel B), colonies of multiple keratinocytes increased in size (arrow) and became walled off by fibroblasts (*). At 10 d post—incubation (panel C), larger colonies of stratified keratinocytes (arrow) aggregated on the plate with evidence of cellular stacks above a basal cell layer. Pictures represent results of 1 of 5 experiments performed in triplicate.

Colony unit formation (cfu) by primary (panel A) and second-passage (panel B) coronary region keratinocytes after 2, 6, and 10 d in coculture. Epidermal keratinocytes (K) isolated directly from coronary region epidermis or after the second passage in vitro were seeded onto confluent monolayers of dermal fibroblasts (F) at 5.0×10³ (low K:F), 7.5×10³ (med K:F), and 1.0×10⁴ (high K:F) cells/cm². ^a,bLetters represent differences (P<0.05) within cell density across time. Data presented as mean±SEM for 5 experiments performed in triplicate.

Expression of IL-1α, IL-1β, IL-1 receptor type I (IL-1RTI), IL-I receptor type II (IL-1RTII), and IL-1 receptor antagonist (IL-1RA) mRNA (panel A); granulocyte-macrophage colony stimulating factor (GM-CSF), keratinocyte growth factor (KGF), and KGF receptor (KGFR) mRNA (panel B); and transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α) mRNA (panel C) in keratinocyte-fibroblast coculture. Cocultures of coronary region keratinocytes and embryonic fibroblasts were cocultured for 12, 24, 48, and 144h. Concentrations of mRNA were determined by real-time PCR, normalized against ubiquitin, and converted to relative expression as arbitrary units (AU) compared with time 0. Data are presented as mean±SEM of 4 experiments performed in duplicate. ^a—cLetters represent significance (P<0.05) across time within gene.