Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2008
Publication Date: 5/10/2009
Citation: Zarlenga, D.S., Mills, J., Dyer, R. 2009. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW. Journal of Dairy Science. 92:1913-1923. Interpretive Summary: Lesions of the hind limb claw horn are the dominant cause of bovine lameness. The lack of knowledge about biologic events that orchestrate normal growth, differentiation and maturation of hoof horn tissue creates challenges given that no in vitro system exists for understanding hoof horn production and its relation to overall animal health. Cell culture models can provide mechanisms by which factors regulating keratinocyte growth and development during normal and abnormal hoof horn growth can be studied. To this end, we established an in vitro culture system of bovine coronette keratinocytes and utilized this system to ask three questions: 1) Do keratinocyte stem cells exist within keratinocyte populations recovered from the bovine claw? 2) Are these fibroblast cells required to support stem cell growth and colony formation? and 3) Are cell-to-cell interactions during keratinocyte stem cell growth accompanied by changes in the expression of immune related genes? The success in developing an in vitro system allowed us to answer these questions and generated important information on early detection signs of lameness and established a model to study methods of treatment.
Technical Abstract: Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocytes and corium fibroblasts, and then employ this system to determine whether or not keratinocytes with stem cell qualities exist in the epidermis of the coronette. Herein, we showed corium fibroblasts were necessary and sufficient to support coronette keratinocyte stem cell colony formation and growth and that such development occurred in association with transcriptional changes in specific cytokine, growth factor and receptor genes when in co-culture. Fibroblasts and coronette keratinocytes from the lateral, hind limb claw were collected and 5.0 x 103 and 7.5 x 103 keratinocytes were cultured in the absence of fibroblasts, or seeded onto bovine dermal fibroblast monolayers. Keratinocytes grown in the absence of dermal fibroblasts formed no colonies while those cultured in the presence of dermal fibroblasts formed 144 ± 15.8, and 183 ± 26.9 keratinocyte colonies, respectively. Keratinocyte stem cell frequency within the primary keratinocytes was estimated at 1.09% (± 0.16) - 1.77% (± 0.28). Quantitative real time PCR showed an increase in mRNA levels for growth factors KGF and GM-CSF, cytokine IL-1, TNF-alpha, and TGF-beta and receptors IL-1RTI, IL-1RTII, IL-1RA in keratinocyte–fibroblast co-cultures. These findings demonstrated that bovine coronette keratinocyte stem cells formed colonies in vitro with an absolute dependence on corium fibroblasts for growth and suggest that germinal hoof horn producing keratinocytes must interact with corium derived fibroblasts to elicit growth of hoof horn tissue.