Establishment of intestinal organoid cultures modeling injuryassociated epithelial regeneration [scRNA-seq]

The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyperorganoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair. Overall design: Examination of single cell mRNA expression pattern of intestine organoids with different conditions in vitro and mouse crypts with different treatments in vivo.

3D类器官（3D organoid）具备模拟生理组织结构与功能的特性，为体外构建发育与疾病模型提供了极为宝贵的研究工具。然而传统类器官培养主要呈现自组织稳态过程，其所用的条件培养基包含VPA、EPZ6438、LDN193189及R-Spondin 1，该培养基可模拟损伤后诱导增生性隐窝形成的肠道上皮再生过程；因此这类类器官被命名为增生性肠道类器官（Hyperorganoids）。单细胞RNA测序（single-cell RNA sequencing）鉴定发现，本研究中的增生类器官内存在多类不同的再生干细胞群，这些细胞群与体内损伤应答性Lgr5+干细胞或Clu+复苏干细胞共享分子特征。进一步分析显示，VPA与EPZ6438对增生类器官的表观基因组重编程及再生过程不可或缺，二者通过表观遗传调控YAP信号通路发挥功能。此外，VPA与EPZ6438可在体内损伤状态下协同促进肠道再生应答。综上，本研究结果证实了一种全新的体外类器官模型，可用于研究上皮再生过程，凸显了引领组织修复的表观遗传重编程的重要性。总体实验设计：对体外不同培养条件下的肠道类器官，以及体内经不同处理的小鼠隐窝，开展单细胞mRNA表达模式检测。