Modelling Sorsby’s Fundus Dystrophy using patient-derived iPSC-RPE

Dewing, Jennifer, Christensen, David RG, Hongisto, Heidi, Scott, Jennifer, Jenkins, Ben, Cree, Angela, Skottman, Heli, Ratnayaka, J. Arjuna and Lotery, Andrew (2019) Modelling Sorsby’s Fundus Dystrophy using patient-derived iPSC-RPE. Investigative Ophthalmology & Visual Science, 60.

Abstract

Purpose : Sorsby’s fundus dystrophy (SFD) leads to bilateral loss of central vision and is caused by mutations in the gene TIMP3. The mechanisms by which TIMP3 mutations cause SFD are poorly understood. iPSC-retinal pigmented epithelial (RPE) cells were generated from SFD patients and controls (ctrl) and cultured on transwell inserts to develop a RPE cell model which was used to study effects of specific TIMP3 mutations on cellular structure and physiology.
Methods : Human fibroblasts were isolated from SFD (TIMP3 S204C) and ctrl subject skin biopsies by explant migration and reprogrammed into iPSCs. These iPSC colonies were cultured in RPE differentiation medium for 4 weeks until sufficient pigmentation was achieved before being transferred to transwells. Transmission electron microscopy (TEM) was used to analyse morphological differences between SFD and ctrl RPE cells. Transepithelial electrical resistance (TEER) was used to measure RPE barrier integrity. To assess the ability of TIMP3 to inhibit MMP activity, protein lysates from SFD and ctrl RPE cells were tested in an EnzCheck collagenase assay.
Results : iPSC-RPE cells were successfully derived from SFD and ctrl patients. TEM analysis revealed significant differences in RPE containing mutant TIMP3 with respect to cell height (SFD 10.3µm ±0.23 SEM, ctrl 13.23µm ±0.43 SEM, p<0.0001) and basal laminar area (SFD 6µm2 ± 0.446 SEM, ctrl 14µm2 ±0.65 SEM, p<0.0001). Striated sub RPE deposits were observed, consistent with long-spacing collagen. A significantly higher number of focal collagen deposits were seen per cell in ctrl-RPE compared to SFD-RPE (SFD 1.444 ±0.44 SEM, ctrl 3.286 ± 0.52 SEM, p<0.05). The number of melanosomes per cell was also significantly higher in ctrl-RPE compared to SFD-RPE (SFD 40.2 ±2.34 SEM, ctrl 57.54 ± 3.74 SEM, p<0.001). However, there was no difference in their basal/apical distribution. SFD-RPE cells showed significantly lower average TEER over 90 days in culture compared to ctrl-RPE (SFD1 161.1 ± 15.9 SEM, ctrl 499.4 ± 49.79 SEM, p<0.001). The enzyme assay showed TIMP3 S204C mutation does not significantly affect the ability of TIMP3 to inhibit collagenase.
Conclusions : The S204C TIMP3 mutation reduces the barrier integrity of RPE cells and results in key morphological alterations to the RPE, including cell height, not previously described. However, our findings indicate that the S204C mutation does not affect TIMP3’s ability to inhibit MMP-9 activity.

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Contributors

Author: Angela Cree

Author: J. Arjuna Ratnayaka

Author: Andrew Lotery

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