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<p>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 (<i>P</i> &lt; 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.</p> Graphical Abstract <p></p>

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Transcriptome Profiling of Trabecular Meshwork Progenitor Cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月27日

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  • Xiaochen Fan,
  • Stephanie Kennedy,
  • Emine K Bilir,
  • Brian Lane,
  • Olivia A Kingston,
  • Xu Chen,
  • Victoria R Kearns,
  • Colin E Willoughby,
  • Carl M Sheridan

摘要

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.

Graphical Abstract