Identification of Quiescent LGR5+ Stem Cells in the Human Colon
Identification of Quiescent LGR5+ Stem Cells in the Human Colon
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DOI：
10.1053/j.gastro.2022.07.081
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发表时间：
2022-10-20
期刊：
影响因子：
29.4
通讯作者：
Sato, Toshiro
中科院分区：
文献类型：
--
作者：
Ishikawa, Keiko;Sugimoto, Shinya;Sato, Toshiro
背景与目的：在小鼠肠上皮中，Lgr5（+）干细胞由于其主要的循环性质而容易受到损伤，其后代去分化以补充干细胞库。然而，人类结肠干细胞在体内平衡和再生过程中如何表现仍不清楚。方法：通过对人和小鼠结肠上皮细胞的单细胞RNA测序分析，分析结肠上皮细胞间的转录异质性。为了追踪人类结肠干细胞或分化细胞的命运，我们通过基因组工程产生了LGR5-tdTomato、LGR5-iCasase9-tdTomato、LGR5-split-Cre和KRT20-ERCreER敲入的人类结肠类器官。用p27- mvenus报告细胞进一步观察p27（+）休眠细胞。为了分析人结肠干细胞在体内的动态，我们将荧光标记的人结肠类器官原位异种移植到免疫缺陷小鼠体内。采用5-乙基-2'-脱氧尿苷脉冲追踪分析评估异种移植细胞的细胞周期动力学。在稳态、LGR5消融和5-氟尿嘧啶诱导的粘膜损伤的背景下，分析了慢循环人干细胞或分化细胞的克隆生成能力。结果：单细胞RNA测序分析阐明了人类结肠中存在非分裂LGR5（+）干细胞。慢循环LGR5(+)p27（+）细胞和原位异种移植的可视化和谱系追踪证实了它们在体内的稳态谱系形成能力，这种能力通过5- fu诱导的粘膜损伤而增强。转化生长因子- β信号调节LGR5（+）细胞的静止状态。尽管分化的KRT20（+）细胞具有可塑性，但它们在5- fu诱导的损伤后没有表现出克隆生长，这表明LGR5(+) p27（+）细胞对生态位环境的占领阻止了邻近分化细胞的去分化。结论：我们的研究结果强调了人类LGR5（+）结肠干细胞的静态特性及其对损伤后再生的贡献。
BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5(+) stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies dedifferentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27(+) dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immunedeficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5(+) stem cells in the human colon. Visualization and lineage tracing of slowcycling LGR5(+)p27(+) cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-beta signaling regulated the quiescent state of LGR5(+) cells. Despite the plasticity of differentiated KRT20(+) cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5(+) p27(+) cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5(+) colonic stem cells and their contribution to post-injury regeneration.