Establishment and transcriptomic characterization of canine organoids from multiple tissues.

Introduction: Organoids are 3-dimensional (3D) stem cell-derived cultures that offer a variety of technical advantages compared to traditional 2-dimensional (2D) cell cultures. Although murine models have proved useful in biomedical research, rodent models often fail to adequately mimic human physiology and disease progression, resulting in poor preclinical prediction of therapeutic drug efficacy and toxicity. An interesting alternative is to use the canine model in research, due to its numerous similarities to humans (shared environment, intact immune system, and development of civilization diseases). The use of canine organoids in drug testing and disease modeling has been limited by the number of models as well as the depth of characterization. Therefore, we believe these types of models can expedite drug testing and create a platform for personalized medicine. Methods: Here, we report the establishment, maintenance, and molecular characterization of six adult-stem cell-derived canine organoid cell lines including endometrium, pancreas, urinary bladder, kidney, lung, and liver from two genetically related canines (B816 and B818). Characterization of these lines was done using multiple techniques including immunohistochemistry (UPKIII, TTF-1) and bulk RNA-seq. Furthermore, scRNA-seq was utilized on a subset of the organoids to identify organoid specific transcriptomic signatures including lung, pancreas, kidney, and bladder. Results: In total, six tissues and organoid lines from each donor were characterized, allowing for a unique, multi-organ comparison between these two individuals and identification of specific cell types within the organoids. Bulk RNA-seq revealed tissue-specific transcriptomic profiles, with organoids enriched in proliferation-related genes and tissues enriched in inflammation-related genes. Principal component analysis showed organ-based clustering, while scRNA-seq identified diverse epithelial subtypes. Conclusion: These organoids begin to establish a platform for reverse translational research, reducing reliance on live animal testing. By leveraging genetically related donors, it highlights tissue-specific variations, facilitating applications in personalized medicine, disease modeling, and pharmacology to bridge veterinary and human research gaps.