Characteristic evaluation of in vitro 3D cell culture model in a multi-dimensional way with high-throughput sequencing

Organoids culture provides unique opportunities to study human diseases and to complement animal models. Several organs and tissues can be in vitro cultured in 3D structures resembling in vivo tissue organization. Organoids culture contains most of the cell types of the original tissue and are maintained by growth factors similar to the in vivo situation. However, the system is yet not fully understood, and specific in vivo features especially those driven by cell-extrinsic factors may be lost in culture. Here we show a comprehensive transcriptome-wide characterization of mouse gut organoids derived from different intestinal compartments and from mice of different gender and age. RNA-seq analysis showed that the in vitro culture strongly influences the global transcriptome of the intestinal epithelial cells (~60% of the total variance). Several compartment-, age- and gender-related transcriptional programs being lost after culturing suggest that they are driven by niche or systemic factors. However, some of the transcriptional programs proper of the cells of origin are kept in vitro indicating that compartment-, age- and gender-specific intrinsic features exist and can be studied in this system. Our study provides knowledge about the cell-extrinsic or cell-intrinsic origin of intestinal epithelial transcriptional programs. We anticipated that our characterization of this in vitro system is an important reference for scientists and clinicians using intestinal organoids as a research model. Bulk RNA sequencing on epithelial cells of small intestine of different compartments from mice of different gender and age in vivo (crypts) and in vitro (organoids)类器官培养为人类疾病研究提供了独特机遇，同时可作为动物模型的重要补充。多种器官与组织可在体外培养为模拟体内组织架构的三维结构。类器官培养体系可保留原组织的绝大多数细胞类型，并通过模拟体内环境的生长因子完成维持培养。然而，该培养体系尚未被完全阐明，部分特定的体内特征——尤其是由细胞外因素驱动的特征——可能会在培养过程中丢失。 本研究对源自不同肠道区域、不同性别与年龄小鼠的小鼠肠道类器官开展了全面的全转录组特征分析。RNA测序（RNA-seq）分析显示，体外培养对肠道上皮细胞的全局转录组具有显著影响，其贡献约占总变异的60%。部分与肠道区域、年龄及性别相关的转录程序在培养后丢失，表明这些程序由微环境（niche）或全身性因素所调控。然而，部分源自起源细胞的固有转录程序在体外培养中得以保留，这表明肠道上皮细胞存在区域、年龄及性别特异性的内在特征，且可通过该培养体系开展相关研究。 本研究阐明了肠道上皮细胞转录程序的细胞外及细胞内在起源机制。我们预期本研究对该体外培养体系的特征分析，可为以肠道类器官为研究模型的科研人员与临床工作者提供重要参考依据。 本研究针对不同性别与年龄小鼠体内（隐窝（crypts））及体外（类器官）来源的不同肠道区域小肠上皮细胞，开展了批量RNA测序（bulk RNA sequencing）。