Invitro modeling of Optic Disc Drusen (ODD) using iPSC-derived retinal cells

Abstract

Purpose : Optic disc drusen (ODD) are acellular refractive calcified deposits in the optic nerve head, which are the most common risk factor for young onset anterior ischemic optic neuropathy (AION). The pathogenesis of ODD is unknown. This study aims to analyze human skin fibroblasts to better understand the molecular events leading to drusen formation and optic neuropathy.

Methods : We consented and prospectively recruited 73 patients with ODD and related optic neuropathies, and healthy controls to collect skin punch biopsies. To assess energy metabolism in ODD patients, we measured P0 skin fibroblast mitochondrial respiration or oxygen consumption rate (OCR) and glycolytic activity (Seahorse Analyzer, Agilent). We then reprogrammed fibroblasts to generate induced pluripotent stem cells (iPSCs) using StemRNA Kits and differentiated to retinal neurons using 2D and 3D retinal organoid models. We analyzed stem cell and retinal neuronal markers and perform multiomic analyses.

Results : Patients fibroblast with autosomal dominant (N=11), sporadic ODD (N=9) and controls. revealed evidence of mitochondrial stress, including significantly reduced basal respiration (P<0.01). iPSCs were derived successfully from patients' fibroblast and characterized for pluripotency markers (Oct3/4, SSEA4, Sox2); and trilineage differentiation potential (Pax6, Brachyury, Sox17 resp). iPSCs were differentiated to RGC using the 2D method, intermediate progenitor cells expressed (Pax6, Nestin) and RGC expressed (Brn3b, TUBB3) by Day45. iPSCs 3D retinal organoids expressed different retinal markers, confirmed by IF at days 40 and 90 (Chx10, Pax6, Brn3B, opsin, recoverin resp). Molecular analysis of iPSC-derived retinal neurons showed differential expression of genes and proteins associated with calcification, mitochondrial biogenesis, endoplasmic reticulum stress, apoptosis. Compared with controls, iPSC-derived retinal neurons exhibited compromised metabolism upon exposure to cellular stress and hypoxia.

Conclusions :
We developed in vitro cellular models of ODD using skin fibroblasts from patients with autosomal dominant and sporadic ODD. These fibroblast-derived iPSC-retinal neuronal 2D and 3D cultures will serve as important tools to elucidate key molecular changes leading to ODD formation and for screening of novel therapies.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.