Establishment and characterization of a canine keratinocyte organoid culture system

BackgroundPerturbations of epidermal and follicular homeostasis have been attributed to a variety of skin diseases affecting dogs. The availability of an invitro system to investigate these diseases is important to understand underlying pathomechanisms. ObjectivesTo establish an accurate and reliable invitro 3D system of canine keratinocyte organoids to lay the basis for studying functional defects in interfollicular epidermis (IFE) and hair follicle (HF) morphogenesis, reconstitution and differentiation that lead to alopecic and epidermal diseases. AnimalsSkin biopsies were obtained from freshly euthanized dogs of different breeds with no skin abnormalities. MethodsCells derived from microdissected IFE and HFs were seeded in Matrigel and keratinocyte organoids were grown and characterized using immunohistochemistry, RT-qPCR and RNA sequencing. ResultsBoth organoid lines develop into a basal IFE-like cell type. Gene and protein expression analysis revealed high mRNA and protein levels of keratins 5 and 14, IFE differentiation markers and intercellular molecules. Key markers of HF stem cells were lacking. Withdrawal of growth factors resulted in upregulation of markers such as KRT16, Involucrin, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. Conclusion and clinical importanceOur 3D invitro culture system provides the basis to explore epidermal function, to investigate the culture conditions necessary for the development of organoids with a HF signature and to address cutaneous disorders in dogs. However, for induction of HF signatures or hair growth, addition of different growth factors or co-culture with dermal papilla will be required.