Table 5_Establishment and transcriptomic characterization of canine organoids from multiple tissues.xlsx

IntroductionOrganoids 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.
MethodsHere, 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.
ResultsIn 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.
ConclusionThese 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.

1. 引言
类器官（Organoids）是源自干细胞的三维（3D）培养体系，相较于传统二维（2D）细胞培养，具备诸多技术优势。尽管啮齿类动物模型已在生物医学研究中得到广泛应用，但这类模型往往无法充分模拟人类生理状态与疾病进程，导致治疗药物的疗效与毒性的临床前预测准确性欠佳。颇具前景的替代方案是在研究中使用犬类模型，因其与人类存在诸多共性：共享生活环境、拥有完整免疫系统，且会罹患与人类类似的文明病。但目前犬类类器官在药物测试与疾病建模中的应用，受限于模型数量与表征深度不足。因此我们认为，此类模型可加速药物测试进程，并为个性化医疗构建研究平台。

2. 方法
本研究报道了源自两只遗传相关犬只（B816与B818）的6株成体干细胞来源的犬类类器官细胞系的构建、培养维持与分子表征，涵盖子宫内膜、胰腺、膀胱、肾脏、肺与肝脏组织。本研究通过免疫组织化学（immunohistochemistry，UPKIII、TTF-1）与批量RNA测序（bulk RNA-seq）等多种技术完成了细胞系的表征。此外，我们还对部分类器官开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），以鉴定肺、胰腺、肾脏与膀胱等组织的类器官特异性转录组特征。

3. 结果
本研究共完成了每只供体犬的6种组织及对应类器官系的表征，实现了两只个体间独特的多器官对比分析，并鉴定出类器官内的特异性细胞类型。批量RNA测序结果显示，各类组织具备组织特异性转录组特征：类器官中增殖相关基因富集，而原代组织中炎症相关基因富集。主成分分析（principal component analysis, PCA）呈现出基于器官的聚类结果，而单细胞RNA测序则鉴定出多种上皮细胞亚型。

4. 结论
本研究构建的类器官为反向转化研究搭建了平台，可减少对活体动物实验的依赖。通过利用遗传相关的供体犬，本研究凸显了组织特异性差异，有助于推动其在个性化医疗、疾病建模与药理学领域的应用，进而弥合兽医与人类医学研究间的鸿沟。