Defining the Activated Fibroblast Population in Lung Fibrosis Using Single Cell Sequencing

In brief, we have applied a novel and unbiased approach to identify distinct and dynamic cell populations, including an in-depth analysis of the fibroblast population, in lung fibrosis. We found that while fibroblast number did not increase after injury, a population of activated fibroblasts can be identified in the fibrotic mouse lung. Using expression profiles of 1,945 lung fibroblasts we redefine the molecular characteristics of activated fibroblasts, finding that they are not a separate population but rather exhibit a similar, yet amplified, gene expression pattern to non-activated cells. A bolus of Bleomycin (2.8 U/kilogram (kg); 1.75 milligram (mg)/kg) or 0.9% sterile saline at 2ml/kg volume was dosed into mouth of each C57BL/6 male mouse (12-13 weeks) near the larynx to induce lung fibrosis. 3 Groups: Saline Control, Bleomycin, and Bleomycin+60mg/kg Nintedanib treatment was started concomitantly with the administration of bleomycin and single cell isolations were performed on day 11.

简言之，本研究针对肺纤维化场景，采用新颖且无偏的方法识别异质性动态细胞群，并对成纤维细胞（fibroblast）群开展了深入分析。研究发现，尽管损伤后成纤维细胞总数未出现显著增加，但在纤维化小鼠肺组织中可鉴定出活化成纤维细胞群。本研究基于1945个肺成纤维细胞的基因表达谱，重新界定了活化成纤维细胞的分子特征：活化成纤维细胞并非独立的细胞亚群，其基因表达模式与非活化成纤维细胞高度相似，仅存在表达水平的扩增上调。本研究通过喉部附近口腔给药的方式，向12~13周龄的C57BL/6雄性小鼠给予单次推注剂量的博莱霉素（Bleomycin，2.8单位/千克（kg），等效于1.75毫克/千克（mg））或0.9%无菌生理盐水，给药体积统一为2ml/kg，以构建肺纤维化模型。实验共设3组：生理盐水对照组、博莱霉素模型组，以及博莱霉素联合60mg/kg尼达尼布（Nintedanib）治疗组；治疗于博莱霉素给药的同时启动，并于造模第11天完成单细胞分离。