Chronic stress promotes intestinal stem cell aging [scRNA-seq]

To elucidate alterations in intestinal cell types under chronic stress, we conducted scRNA-seq analysis of intestinal crypts from NT and ES models. After that, we found that significant changes in ISCs in the ES group compared to the NT group. To validate the functional roles of ISCs, we performed RNA-seq of ISCs under different treatment conditions, we identified Chrm3-dependent differential genes between NT and ES groups, particularly noting downregulated genes associated with stemness and proliferation (e.g., Olfm4, Lgr5, and Mcm4), and upregulated genes linked to aging and calcium signaling pathways (e.g., Cdkn1a, Orai1, and Chp2), which contribute to ISC aging. These findings provided mechanistic insights into targeting these pathways to enhance intestinal function and integrity. Furthermore, to assess the impact of stress-induced changes in microbiota composition on ISC stemness, we synchronized microbiota between NT and ES groups through co-housing conditions and employed 16S rDNA sequencing. This analysis aimed to ascertain the possibility that changes in the microbiota composition whether contribute to the decline in ISC stemness under stress conditions. scRNA-seq of crypts were used to to characterize the diversity of cell lines under chronic stress. RNA-seq of ISC in Chrm3Lgr5+/+ and Chrm3Lgr5-/- mice from NT and ES mice were taken to delineate altered pathways and the mechanisms underlying ISC changes in ES model. 16S rDNA-seq (available in PRJNA1090629) were employed to confirm microbiota synchronization between NT and ES groups under co-housing conditions. Overall design: Crypts from wild-type mice in the NT and ES groups were isolated and analyzed using single-cell RNA sequencing (scRNA-seq).

为阐明慢性应激下肠道细胞类型的动态变化，我们对NT组与ES模型小鼠的肠道隐窝开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析。后续分析发现，相较于NT组，ES组的肠干细胞（intestinal stem cells, ISCs）存在显著改变。
为验证ISCs的功能作用，我们针对不同处理条件下的ISCs开展了RNA测序（RNA-seq）分析，进而鉴定出NT组与ES组之间存在Chrm3依赖的差异表达基因。其中，与干细胞干性及增殖相关的基因显著下调（如Olfm4、Lgr5及Mcm4），而与衰老及钙信号通路相关的基因则显著上调（如Cdkn1a、Orai1及Chp2），上述变化共同参与了ISCs的衰老过程。
本研究结果为靶向上述通路以改善肠道功能与完整性提供了机制层面的理论依据。
此外，为探究应激诱导的菌群组成改变对ISCs干性的影响，我们通过同笼饲养策略实现了NT组与ES组小鼠肠道菌群的同步化，并采用16S核糖体DNA（16S rDNA）测序技术开展相关分析。
本分析旨在明确应激条件下菌群组成的改变是否会导致ISCs干性水平下降。
我们还对肠道隐窝开展了scRNA-seq分析，以解析慢性应激下细胞谱系的多样性。
我们还获取了NT组与ES组小鼠中Chrm3Lgr5+/+及Chrm3Lgr5-/-小鼠的ISCs RNA-seq数据，以描绘ES模型中ISCs异常改变所涉及的通路及潜在分子机制。
16S rDNA测序数据（登录号PRJNA1090629）已公开可获取，用于验证同笼饲养条件下NT组与ES组小鼠的肠道菌群同步化效果。
整体实验设计：分离NT组与ES组野生型小鼠的肠道隐窝，采用单细胞RNA测序（scRNA-seq）进行分析。