Reduced cystatin C secretion in iPS-derived retinal pigment epithelium cells is associated with altered extracellular matrix adhesion and migration

Abstract

Purpose : The protease inhibitor cystatin C is one of the most abundantly expressed proteins in retinal pigment epithelium (RPE) cells. A single point mutation, leading to an A25T amino acid substitution in the leader sequence, is associated as a recessive variant with reduced cystatin C secretion and is a known risk factor for developing age-related macular degeneration (AMD). This study tested the hypothesis that reduced cystatin C secretion is associated with functional changes in RPE migration and adhesion when cultured on selected extracellular matrix (ECM) proteins.

Methods : Bi-allelic CRISPR/Cas9-mediated gene editing was employed to generate induced pluripotent stem cells (iPSCs) expressing the A25T mutant variant of cystatin C, which were then differentiated to RPE cells. Cellular migration and adhesion of two independent iPS-RPE clones were tested by wound healing assay after 8 weeks of culture on matrigel, laminin, collagen IV or fibronectin (n=4), and cell adhesion assay when seeded on the same ECM proteins (n=3). NIH3T3 fibroblasts were grown on matrigel in the presence of fluorescently labelled laminin or fibronectin to incorporate these ECM proteins into the deposited 3D-matrix, followed by gentle lysis with 20 mM NH₄OH. Fluorescence microscopy was then used to assay the degradation of laminin or fibronectin (n=3) in the decellularised matrix. Statistical analyses were performed using Student’s two-tailed t-test.

Results : Cystatin C gene edited iPS-RPE cells had notable disruption of cystatin C secretion, confirmed by immunoblotting, and displayed significantly higher rates of migration 7 days post wound infliction when cultured on matrigel, collagen IV or fibronectin, compared with wild type iPS-RPE cells (p<0.05). Adhesion of edited cells was also significantly faster when seeded on collagen IV or fibronectin (p<0.05), but not matrigel or laminin. Furthermore, gene edited cells showed a higher rate of laminin and fibronectin protein degradation 3 days after seeding on fluorescently labelled proteins incorporated into matrigel (p<0.05).

Conclusions : Disruption of cystatin C secretion in RPE tissue leads to altered ability for cells to adhere and migrate on ECM, as well as a higher rate of ECM protein degradation. All these effects have potential relevance for AMD pathogenesis.

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