Characterization of Stargardt disease patient-derived human retinal organoids harboring the p.Gly1961Glu mutation in the ABCA4 gene

Abstract

Purpose : To investigate the cellular pathophysiology of Stargardt disease caused by the c.5882G>A p.(Gly1961Glu) in a patient-derived retinal organoid model system.

Methods : Human induced pluripotent stem cells (iPSCs) were reprogrammed from peripheral blood lymphocytes from 4 STGD1 patients and 2 two unaffected control subjects. One patient was homozygous for the p.Gly1961Glu variant, two were compound heterozygous, and one patient harbored two deleterious ABCA4 alleles. Mycoplasma testing and karyotyping was performed before differentiation, and pluripotent markers, LIN28, NANOG, OCT4, and SOX2 expression were confirmed by PCR. To begin differentiation, hiPSCs were cultured in neuron-inducing medium for 16 days and then switched to retina-inducing medium. At day 42, 10% FBS and 100uM taurine were added to the medium for long-term differentiation. Developing organoids were manually picked between days 25 and 30, and cultured in suspension. Cryosectioning and immunofluorescence (IF) microscopy were performed at days 90, 120,150, and 230, followed by transmission electron microscopy (TEM) at day 260.

Results : At day 230, organoids from one compound heterozygous patient for the p.(Gly1961Glu) and c.4947del p.(Glu1650fs) variants, exhibited round contours with visible lamination of the neural retina. Rudimentary subcellular compartments of photoreceptors along the outer edge were discernible on IF by Hoeschst (nuclei), RHO (rods) and Opsin (Blue, Red/Green cones) staining. These distal structures were visualized by TEM and are consistent with short, nascent outer segments. Mutant ABCA4 protein was detected using two separate antibodies--ABCA4 RIM3F4 and RIM5B4, and co-localized with Opsin Blue, or Opsin Red/Green in photoreceptors.

Conclusions : Patient-derived retinal organoids recapitulate complex cellular features of the human retina and express mutant ABCA4 protein in the distal portions of developing photoreceptors. These findings provide a functional model system to characterize the physiological consequences of ABCA4 mutations in individual patients and simultaneously offers a broad window for understanding the mechanism of retinal degenerative diseases.

This is a 2021 ARVO Annual Meeting abstract.