Establishing induced pluripotent stem cell lines from two dominant optic atrophy patients with distinct OPA1 mutations and clinical pathologies

Abstract

Dominant optic atrophy (DOA) is an inherited disease that leads to the loss of retinal ganglion cells (RGCs), the projection neurons that relay visual information from the retina to the brain through the optic nerve. The majority of DOA cases can be attributed to mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial-targeted protein that plays important roles in maintaining mitochondrial structure, dynamics, and bioenergetics. Although OPA1 is ubiquitously expressed in all human tissues, RGCs appear to be the primary cell type affected by OPA1 mutations. DOA has not been extensively studied in human RGCs due to the general unavailability of retinal tissues. However, recent advances in stem cell biology have made it possible to produce human RGCs from pluripotent stem cells (PSCs). To aid in establishing DOA disease models based on human PSC-derived RGCs, we have generated iPSC lines from two DOA patients who carry distinct OPA1 mutations and present very different disease symptoms. Studies using these OPA1 mutant RGCs can be correlated with clinical features in the patients to provide insights into DOA disease mechanisms.

Keywords: DOA; OPA1; RGC; dominant optic atrophy; induced pluripotent stem cells (iPSC); retinal ganglion cell.

FIGURE 1

Fundus imaging and visual field testing. (A) Fundus images of Patient 1 and Patient 2 displaying bilateral, temporal optic nerve head pallor. (B) Humphrey visual field (HVF) testing reports. Patient 1’s HVF 30-2 showed bilateral, temporal, paracentral scotomas. Patient 2’s HVFs detected small paracentral scotomas. OD: oculus dexter, right eye; OS: oculus sinister, left eye.

FIGURE 2

Retinal lamination imaging by optical coherence tomography. (A) Patient 1’s OCT results demonstrate bilateral retinal nerve fiber layer (RNFL) thinning, most significantly in the temporal zone, involving both superior and inferior regions. (B) Patient 2’s OCT results reveal bilateral, inferior RNFL thinning, accompanied by some superior fiber loss in both retinas. ILM, inner limiting membrane; RNFL, retinal nerve fiber layer; TMP (T); temporal; SUP (S): superior; INF (I): inferior; NAS (N): nasal.

FIGURE 3

Characterization of OPA1-mutant iPSCs from DOA patients. (A) Brightfield images of iPSCs reprogrammed from DOA Patient 1 (1iDOA) and Patient 2 (2iDOA) display normal pluripotent stem cell morphology (top row) and stain positive for alkaline phosphatase (bottom row). Scale bars: 200 μm (top row), 500 μm (bottom row). (B) 1iDOA and 2iDOA iPSCs express the pluripotent stem cell markers SOX2, OCT3/4, and NANOG similarly to the wild type ESC line, UCLA1. Scale bar: 40 μm. (C) Both iPSC lines display a 46, XY normal male karyotype. (D) DNA sequences of iPSC lines 1iDOA and 2iDOA (bottom row) aligned to the corresponding regions of OPA1 in the wild type (WT) reference genome (top row). Sequences confirm that 1iDOA contains a heterozygous, single base pair (G) insertion in exon 19 and that 2iDOA has a heterozygous, 2 base pair (AT) deletion in exon 13. Peaks reflect both WT and Mutant allele bases at a given position. Allele sequence identities are clarified above peaks. Boxed regions indicate bases inserted (1iDOA, mutant allele) or deleted (2iDOA, WT allele).

FIGURE 4

Detection of ROS production in PSC lines. Detection of mitochondria and cellular ROS in pluripotent stem cell (PSC) lines were performed using live cells. (A, C, E) show representative signals of ROS (green), MitoTracker (red) and merged images for 1iDOA, 2iDOA, and the control ESC H9 lines. (B, D, F) show merged ROS and MitoTracker signals after the perspective PSC lines were treated with menadione to increase ROS. Scale bar, 20 μm for all.