Fan Xiaochen, Kennedy Stephanie, Bilir Emine K, Lane Brian, Kingston Olivia A, Chen Xu, Kearns Victoria R, Willoughby Colin E, Sheridan Carl M
Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, United Kingdom.
Department of Medicine, University of Cambridge, Cambridge, United Kingdom.
Stem Cell Rev Rep. 2025 May 27. doi: 10.1007/s12015-025-10900-0.
The loss and dysfunction of trabecular meshwork (TM) cells are implicated in aging and primary open-angle glaucoma. TM progenitor cells (TMPCs) contribute to the population and function of the TM, but their identity is not well elucidated. This study aimed to identify the expression profile of differentially expressed genes (DEGs) in human TM cell cultures, TM-derived spheres, and their differentiated progeny. Primary normal human TM cells (PTM) from three donors were cultured, de-differentiated into spheres, and re-differentiated into TM cells (DTM). RNA-Seq was performed using Illumina NGS, and bioinformatics analysis was conducted with Tuxedo, Bowtie2, Tophat, Cufflinks, and Ingenuity Pathway Analysis (IPA). DEGs were validated via Nanostring, RT-qPCR (in five independent donors), immunocytochemistry, and western blotting. RNA-seq identified significant DEGs in PTM, TM progenitor cells (TMPCs), and DTM cells. Gene expression in TMPCs differed significantly from PTM and DTM cells. Nanostring and RT-qPCR confirmed 70 DEGs upregulated in TMPCs (P < 0.05). Immunocytochemistry highlighted distinct markers in TMPCs (SOX2, NOTCH1, ANKG, MGP) versus PTM and DTM cells (TAGLN, TEM7, SPARC). Western blotting further analyzed MGP, TAGLN, and SPARC proteins, revealing significant upregulation of MGP in TMPCs and downregulation of TAGLN and SPARC in spheres compared to PTM cells. Pathway analysis revealed activation of cell cycle checkpoint regulation, SUMOylation, and STAT3 pathways in TMPCs, with HGF, MMP9, KDR, IGF1, and FOS as key node genes in TMPC development. RNA-Seq identified novel expression profile of potential TM markers and activated pathways in TMPCs, providing insights into TMPC behaviours in physiological and pathological conditions.
小梁网（TM）细胞的丢失和功能障碍与衰老及原发性开角型青光眼有关。TM祖细胞（TMPCs）对TM的细胞数量和功能有贡献，但其特性尚未完全阐明。本研究旨在确定人TM细胞培养物、TM来源的球状体及其分化后代中差异表达基因（DEGs）的表达谱。来自三名供体的原代正常人TM细胞（PTM）被培养，去分化为球状体，然后再分化为TM细胞（DTM）。使用Illumina NGS进行RNA测序，并使用Tuxedo、Bowtie2、Tophat、Cufflinks和Ingenuity Pathway Analysis（IPA）进行生物信息学分析。通过Nanostring、RT-qPCR（在五个独立供体中）、免疫细胞化学和蛋白质免疫印迹法对DEGs进行验证。RNA测序确定了PTM、TM祖细胞（TMPCs）和DTM细胞中的显著DEGs。TMPCs中的基因表达与PTM和DTM细胞有显著差异。Nanostring和RT-qPCR证实了TMPCs中70个上调的DEGs（P < 0.05）。免疫细胞化学突出了TMPCs（SOX2、NOTCH1、ANKG、MGP）与PTM和DTM细胞（TAGLN、TEM7、SPARC）中的不同标志物。蛋白质免疫印迹法进一步分析了MGP、TAGLN和SPARC蛋白，显示与PTM细胞相比，TMPCs中MGP显著上调，球状体中TAGLN和SPARC下调。通路分析显示TMPCs中细胞周期检查点调节、SUMO化和STAT3通路被激活，HGF、MMP9、KDR、IGF1和FOS是TMPC发育中的关键节点基因。RNA测序确定了TMPCs中潜在TM标志物的新表达谱和激活的通路，为生理和病理条件下TMPC的行为提供了见解。
