Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis. Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis

We performed transcriptomic studies comparing phenotypically defined PMSCs (PDGFRa/CD34/CD9high:CD200low) to HSCs (Vitamin-A fluorescent) of high purity, using bulk RNAseq. We further extended bulk RNAseq analyses of the two cell populations to myofibroblasts derived from both cell types in culture, including an early and late stage of differentiation for PMSC-MFs. Comparative analyses of gene expression indicated that 100 genes in PMSCs and 112 genes in HSCs, i) were overexpressed according to both scRNAseq and bulk RNAseq analyses, and ii) remained differentially expressed in these cells throughout their myofibroblastic differentiation. We selected the most differentially expressed of these genes, to build a multigene expression signature of PMSCs/PMSC-MFs (7 genes) and of HSCs/HSC-MFs (6 genes). Overall design: mRNA profiles of PMSCs (n=4) and HSCs (n=4), and their derived myofibroblasts in culture, including PMSC-MFs-3d (n=4), PMSC-MFs-7d (n=4) and HSC-MFs-3d (n=4).

本研究采用批量RNA测序（bulk RNAseq），对高纯度的表型鉴定的PMSCs（PDGFRa/CD34/CD9高表达：CD200低表达）与经维生素A荧光标记的HSCs开展转录组学比较分析。本研究进一步将两种细胞群体的批量RNA测序分析拓展至体外培养中由二者分化得到的肌成纤维细胞，其中包含PMSC来源的肌成纤维细胞（PMSC-MFs）的早期与晚期分化阶段。基因表达比较分析结果显示，PMSCs中存在100个基因、HSCs中存在112个基因同时满足以下两项特征：其一，经单细胞RNA测序（scRNAseq）与批量RNA测序（bulk RNAseq）分析均证实其表达上调；其二，在这些细胞的肌成纤维细胞分化全过程中始终保持差异表达。本研究筛选出其中差异表达最为显著的基因，分别构建了PMSCs/PMSC-MFs的多基因表达特征（包含7个基因）与HSCs/HSC-MFs的多基因表达特征（包含6个基因）。整体实验设计如下：检测了PMSCs（n=4）、HSCs（n=4）及其体外培养来源的肌成纤维细胞的mRNA表达谱，其中包括PMSC-MFs-3d（n=4）、PMSC-MFs-7d（n=4）与HSC-MFs-3d（n=4）。