Tissue specific human fibroblast differential expression based on RNAsequencing analysis

Background:Physical forces, such as mechanical stress, are essential for tissue homeostasis and influence geneexpression of cells. In particular, the fibroblast has demonstrated sensitivity to extracellular matrices with assumedadaptation upon various mechanical loads. The purpose of this study was to compare the vocal fold fibroblastgenotype, known for its unique mechanically stressful tissue environment, with cellular counterparts at various otheranatomic locales to identify differences in functional gene expression profiles. Results:By using RNA-seq technology, we identified differentially expressed gene programs (DEseq2) among sevennormal human fibroblast primary cell lines from healthy cadavers, which included: vocal fold, trachea, lung, abdomen,scalp, upper gingiva, and soft palate. Unsupervised gene expression analysis yielded 6216 genes differentially expressedacross all anatomic sites. Hierarchical cluster analysis revealed grouping based on anatomic site origin rather thandonor, suggesting global fibroblast phenotype heterogeneity. Sex and age-related effects were negligible. Functionalenrichment analyses based on separate post-hoc 2-group comparisons revealed several functional themes within thevocal fold fibroblast related to transcription factors for signaling pathways regulating pluripotency of stem cells andextracellular matrix components such as cell signaling, migration, proliferation, and differentiation potential. Conclusions:Human fibroblasts display a phenomenon of global topographic differentiation, which is maintained inisolation via in vitro assays. Epigenetic mechanical influences on vocal fold tissue may play a role in uniquely modellingand maintaining the local environmental cellular niche during homeostasis with vocal fold fibroblasts distinctlyspecialized related to their anatomic positional and developmental origins established during embryogenesis. Overall design: Comppared mRNA profiles of a total of 34 samples from 7 different locations controlling for age and sex effect.

背景：物理力（如机械应力）是维持组织稳态的核心要素，同时可调控细胞的基因表达。其中，成纤维细胞（fibroblast）已被证实对细胞外基质具有敏感性，并可适应多种机械负荷。本研究旨在以具有独特机械应激组织环境的声带成纤维细胞为研究对象，将其基因型与其他不同解剖部位的同源细胞进行比较，以揭示其功能基因表达谱的差异。
结果：本研究通过RNA测序（RNA-seq）技术，利用DEseq2鉴定了来自健康遗体的7株正常人成纤维细胞原代细胞系的差异表达基因程序，这些细胞系分别取自声带、气管、肺、腹部、头皮、上颌牙龈及软腭。经无监督基因表达分析，共鉴定出6216个在所有解剖部位间存在差异表达的基因。层次聚类分析显示，细胞聚类结果依据其解剖部位来源而非供体个体，提示成纤维细胞存在全局的表型异质性。性别与年龄相关的影响可忽略不计。基于两两分组事后比较的功能富集分析结果表明，声带成纤维细胞存在多个功能特征：涉及调控干细胞多能性信号通路的转录因子，以及与细胞信号传导、迁移、增殖及分化潜能相关的细胞外基质组分。
结论：人类成纤维细胞存在全局的位置特异性分化现象，该特性在体外实验体系中可独立维持。表观遗传层面的机械调控作用可能参与塑造并维持声带组织的局部细胞微环境稳态；声带成纤维细胞的独特特化特性与其胚胎发育阶段确立的解剖位置及发育起源密切相关。
实验整体设计：本研究共纳入来自7个不同解剖部位的34份样本，通过控制年龄与性别因素，对比了各样本的mRNA表达谱。