背景:严重的情况下,角膜上皮功能障碍或缺乏会导致视力障碍或失明。角膜上皮细胞的快速有效再生依赖于角膜缘干细胞(LSC)。然而,LSCs及其小生境细胞对损伤的分子和功能反应仍然难以捉摸。
方法:对来自正常小鼠和角膜上皮缺损模型的角膜组织进行单细胞RNA测序。进行生物信息学分析以确认LSC的独特特征和细胞命运。进行了Creb5和OSM治疗实验的击倒以确定它们在角膜上皮伤口愈合中的作用。
结果:我们的数据定义了LSCs的分子特征,并重建了角膜上皮细胞的假时间轨迹。基因网络分析表征了可能调节LSC动力学的转录标志,并鉴定了转录因子Creb5,该转录因子在LSCs中表达并在损伤后显著上调。功能丧失实验表明,沉默Creb5会延迟角膜上皮愈合和LSC动员。通过细胞间通讯分析,我们确定了609个候选再生相关的配体-受体相互作用对LSCs和不同的小生境细胞之间,并在损伤后发现了一个独特的Arg1+巨噬细胞亚群,它们作为制瘤素M(OSM)的来源存在,IL-6家族细胞因子,被证明可以有效地加速角膜上皮伤口的愈合。
结论:本研究为发现眼表重建的机制和潜在的临床干预措施提供了宝贵的单细胞资源和参考。
BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive.
METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing.
RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing.
CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.