SRA

The epidermis and associated appendages ensure a number of critical functions necessary for survival and social interactions. Perturbations of epidermal stem cell homeostasis lead to a variety of skin diseases affecting humans and dogs. Therefore, the establishment of an in vitro system to investigate canine epidermal stem cells, representing a model for human diseases, is essential. Here we report the establishment and characterization of organoids derived from microdissected canine hair follicles (HFs) and interfollicular epidermal (IFE). Gene and protein expression analysis revealed high mRNA and protein levels of keratin 5 and 14, IFE differentiation markers and intercellular molecules (e.g., keratin 10 and desmoglein), indicating a strong basal cell signature as well as differentiation towards mature epidermis. Key markers of HF stem cells were lacking. This suggests that, independently of the tissue of origin, both organoid lines develop into the same cell type (basal IFE-like keratinocytes). Signaling pathways members important for regulation of HF growth and cycling (such as Wnt, Hh, BMP and Notch) were present in both HF and IFE organoids at low levels. Withdrawal of growth factors (Noggin, R-spondin and FGFs) resulted in upregulation of markers such as KRT16, IVL, KRT17 and SOX9, showing the potential of the organoids to develop towards more differentiated tissue. However, for induction of HF signatures or hair growth, addition of different growth factors or dermal papilla co-culture might be required. Taken together, our in vitro culture system can provide the basis to address hair growth and explore epidermal function/regeneration, allowing us to further investigate pathomechanisms of cutaneous disorders in dogs and potentially human patients. Overall design: We establish an organoid culture system for long-term expansion of mouse epidermal stem cells. Using histological methods as well as low-coverage multiplexed RNA sequencing, we show that cultured organoids resembled interfollicular epidermis. We analyzed a total of 23 samples, including 6 controls that are isolated from the skin of dog. None-passaged as well as cultured organoids were compared with replicates. Differences growth factors and small molecules that allow expansion of organoids were compared with replicates.