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Jane Hu, Nermin Kady, Ashley Macabasco, Michael B Gorin, Anna Matynia, Roxana A Radu; Complement Dysregulation is Evidenced in iPSC-derived RPE Cells from Stargardt Disease patients. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1507.
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© ARVO (1962-2015); The Authors (2016-present)
Recessive Stargardt (STGD), a central blinding disease similar to AMD, is caused by mutations in the ABCA4 gene. Both STGD patients and the Abca4-/-, its mouse model, exhibit deposition of bisretinoid-lipofuscin in the retinal pigment epithelium (RPE) and photoreceptors degeneration. Bisretinoid-mediated complement activation was previously reported in cultured RPE cells and detected in the RPE of Abca4-/- mice. In this study, we used induced pluripotent stem cell (iPSC) derived RPE from STGD patients to investigate the role of complement system in disease pathogenesis.
Fibroblasts of STGD patient, with a heterozygous ABCA4 variant (R1129L/C3386G>T), and unaffected control were reprogrammed into iPSCs and differentiated to RPE cells using standard protocols. iPSC-RPE cells were grown on filters and analyzed after 3- and 12-mo by confocal and electron microscopy. Morphometric analysis were implemented to determine the cell count and height. Transepithelial resistance (TER) was measured to evaluate the RPE monolayer integrity. Immunohistochemistry was performed on the flatmount of iPSC-RPE cells with antibodies against ZO1, complement regulator CD46, C3/C3b/iC3b components, and membrane attack complex (MAC).
At 3-mo, both control and STGD iPSC-RPE cells were maintained at similar cell count and developed normal morphology. ZO1 staining and TER measurements were comparable in control and STGD iPSC-RPE cells. Importantly, CD46 levels were reduced in STGD iPSC-RPE by 55% of control cells. In contrast, C3/C3b/iC3b and MAC levels were increased by 39% and 50% respectively in STGD iPSC-RPE vs control cells. At 12-mo, STGD iPSC-RPE cells showed significant reduction in both cell height and number (~30%) vs control cells. These findings correlated with extensive reduction of TER in STGD iPSC-RPE cells (by 80% vs control), reflecting cell membranes disruption. Additionally, the autofluoscence level was about 60% more in STGD vs control iPSC-RPE cells.
Using STGD patient iPSC-derived RPE cells, we provide evidence of complement dysregulation manifested with elevated C3/C3b/iC3b and MAC deposition. Additionally, ongoing bisretinoid-mediated complement activation led to disruption of cellular membrane and age-dependent cell loss in 12-mo cultured STGD iPSC-RPE cells. These findings strongly support a common inflammatory etiology of AMD and STGD maculopathies.
This is a 2020 ARVO Annual Meeting abstract.
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