Characterization of canine epidermal organoid cultures by immunohistochemical analysis and quantitative PCR

Abstract

Background Keratinocyte organoids can be used as a tool to evaluate epidermal structure, function and dysfunction. Objectives To optimize the canine keratinocyte organoid system and produce organoids that are structurally equivalent to in vivo canine epidermis, in order to enable studies that focus on epidermal diseases and diseases resulting from an impaired epidermal barrier. Animals Skin biopsies were obtained from five recently euthanized dogs of different breeds with no skin abnormalities. Methods and materials Cells derived from microdissected interfollicular epidermis were seeded in basement membrane extract and epidermal organoids were grown under different media conditions. Organoids were characterized to assess cell morphology and architecture in haematoxylin and eosin‐stained slides and expression of selected epidermal markers (keratin 5, keratin 10, loricrin and filaggrin) by immunohistochemical analysis and quantitative reverse transcription PCR. Results The selected epidermal markers were expressed in the same epidermal layers in the organoids cultured in expansion medium and differentiation medium as in normal interfollicular epidermis, yet restriction to the distinct layers was best achieved with expansion medium. Comparison of the mRNA expression levels of these markers revealed that relative expression is similar in organoids cultured in expansion medium and normal canine epidermis, while it differs in organoids cultured in differentiation medium. Conclusion and clinical importance Organoids cultured in expansion medium have an equivalent structure to the interfollicular epidermis and express key marker proteins in similar proportions. Epidermal organoids are therefore a promising in vitro model to study epidermal structure, function and dysfunction.

Treatment of Canine Type 1 Diabetes Mellitus: The Long Road from Twice Daily Insulin Injection towards Long-Lasting Cell-Based Therapy

“…Organoids bridge between in vitro and in vivo models, evaluating biological cell functions, drug screening, and cellular differentiation and in addition might be used for cell transplantations (Figure 2). Organoid technology is gaining momentum in veterinary research as indicated by studies performed on organoids in dogs and cats with various organs, like the liver [51][52][53][54], intestine [55][56][57], and skin [58]. Direct reprogramming of stem cells (skin-derived?)…”

Farm and Companion Animal Organoid Models in Translational Research: A Powerful Tool to Bridge the Gap Between Mice and Humans

“…The studies on canine keratinocyte and epidermal organoid culture systems have been reported ( 93 , 94 ). In the study, keratinocyte organoids from both microdissected interfollicular epidermis and hair follicle tissues were successfully established using culture medium containing Noggin, R-spondin-1, and Rho kinase inhibitor ( 93 ).…”

“…The study also demonstrated that Wnt3a was not essential to grow canine keratinocyte organoids, thus was removed from the expansion media. Furthermore, an attempt to optimize the canine keratinocyte organoid system and develop an epidermal organoid revealed that supplementation of Wnt3a and other factors to differentiation medium did not have beneficial effects on inducing differentiation of the interfollicular epidermis-derived organoids closer to normal epidermis ( 94 ).…”

Establishment and Characterization of An HER2-enriched Canine Mammary Cancerous Myoepithelial Cell Line with Stem Cell Properties

“…However, only a few canine organoids have been reported. For instance, a canine prostate cancer organoid and canine bladder cancer organoid were established using urine samples [47,48]; a canine keratinocyte organoid and epidermal organoid were developed utilizing hair follicles [49,50]; canine intestinal organoids were derived from the intestinal stem cells of both healthy and gastrointestinal-diseased dogs [51]. Despite the high morbidity of CMT, the development of a CMT organoid for better understanding the disease process has not been reported.…”