Characterization of gene expression profiles of cultured human primary trabecular meshwork cells, transformed cell lines and human skin fibroblasts

Abstract

Purpose : The trabecular meshwork (TM) has a central role in regulating intraocular pressure (IOP). Dysfunction of the TM may cause high IOP and glaucoma. Characterizing gene expression in primary and transformed TM cells is necessary for better disease modeling and for honing cell replacement strategies (i.e. via induced pluripotent stem cell methods). In this project we determine the gene expression patterns in primary human TM cells and in transformed human TM cells. In addition, we will investigate the mechanism of glaucoma specific mutations (MYOC) in patient-derived fibroblast cells.

Methods : RNA-seq analysis was performed on RNA collected 3 primary TM cell cultures, 3 technical replicates of nTM5 cell and gTM3 cell lines, fibroblast cell cultures form 3 normal subjects and from 3 glaucoma patients with Gly367Arg MYOC mutation. Catalogues of expressed genes were generated by identifying the genes with greater than 200 million reads. Genes were judged to have differential expression between primary & transformed TM cells, and control & patients' fibroblasts if the absolute value of the log2 (fold change) was > 1, the q-value < 0.001.

Results : Each cell type had a unique gene expression profile. The five most highly expressed genes in primary TM cells were FN1, FTH1, VIM, EEF1A1 and FLNA. The five most highly expressed genes in primary TM cells that encoded secreted proteins were: FN1, PSAP, MMP2, CLU and COL1A2. Comparison of gene expression between fibroblast cells from normal subjects and glaucoma patients with a Gly367Arg MYOC mutation identified several differentially expressed genes, including WNT4, LHX9, MMP10, and IL-33. Pathway analyses suggest that MYOC mutations may influence a number of important biological processes including: extracellular matrix organization, wound healing, DNA binding, and transcription.

Conclusions : Gene clusters analyses suggest transformed cell lines and primary TM cell strains share similarities though gTM3 has more resemblance than TM5. Genes that are most highly expressed especially ones encoding secreted proteins in TM cells will be studied in depth to differentiate iPSCs into TM-like cells using modified Taguchi method. iPSC-derived TM like cells from myocilin glaucoma patients skin fibroblasts will aid in studying glaucoma pathogenesis in the most relevant cell type and developing cell replacement therapies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.