Establishment of human Leber's hereditary optic neuropathy model using iPSC-derived retinal organoids

Abstract

Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease caused by mitochondrial DNA mutations, leading to central vision loss and retinal ganglion cell (RGC) degeneration. Progress in understanding LHON and developing treatments has been limited by the lack of human-like models. In this study, we aimed to establish a human retinal model of LHON using retinal organoids (ROs) from LHON patient-derived induced pluripotent stem cells (LHON-iPSCs). We first confirmed LHON-iPSCs were successfully differentiated into ROs (LHON-ROs). LHON-RO showed a reduction in RGC numbers and the density of neural axons. Additionally, both mitochondrial membrane potential and ATP production were decreased in LHON-RO. Finally, treatment with idebenone, the only approved therapeutic agent for LHON, improved RGC numbers in LHON-RO. This model replicates key clinical features of LHON, including RGC and axonal loss, and demonstrates idebenone's therapeutic potential. Furthermore, a comprehensive analysis of the LHON-RO model revealed impaired mitophagy, suggesting novel therapeutic targets for LHON. Thus, the LHON-RO model offers a valuable platform for studying LHON pathogenesis and evaluating treatments.

Keywords: Leber’s hereditary optic neuropathy; in vitro disease modeling; mitochondrial disease; mitophagy; retinal organoid.

Figure 1

Protocol for RO differentiation. (A) Schematic of mitochondrial DNA and mutation points in LHON-iPSCs. Created in BioRender.com. (B) Differentiation protocol for retinal organoids (ROs). ESCM: embryonic stem cell medium, EBM: embryoid body medium, NIM: neural induction medium, RDM: retinal differentiation medium, 3D-RDM: three-dimensional retinal differentiation medium. (C) Staining for retinal ganglion cell (RGC) marker (magenta: Brn3), retinal progenitor cell (RPC) marker (blue: Chx10), and neural marker (green: TUJ1). Scale bars: 100 μm. (D) Quantification of the size of the ROs at Day35. Mean ± SEM (Cont: n = 46, LHON: n = 41).

Figure 2

The number of RGCs and neurons was reduced in LHON-RO. (A,B) The expression levels of mRNA for BRN3A and TUBB were measured by qPCR of ROs at Day 35. Mean ± SEM (Control: n = 6–8, LHON: n = 8–11). Student’s t-test (two-tailed), *p < 0.05 (A: p = 0.045, B: p = 0.042). (C,D) Staining for RGC marker (magenta: Brn3) and neural marker (green: TUJ1) in Day 35 Control-RO and LHON-RO. (C) Whole, (D) slice. Scale bars: 100 μm. (E) The number of RGCs in the entire RO was counted. (F) The number of neurons was counted, and the intensity of TUJ1 expression was measured. Mean ± SEM (Control: n = 6, LHON: n = 9). Student’s t-test (two-tailed), *p < 0.05 (E: p = 0.011), ***p < 0.001. (G) Staining for RGC marker (Red: Brn3) and apoptosis marker (green: TUNEL) in Day 35 Control-RO and LHON-RO. Arrow: Brn3⁺, TUNEL⁺ cells. Scale bars: 100 μm. (H) The percentage of TUNEL-positive RGCs relative to the total number of RGCs was measured. Mean ± SEM (n = 6). Student’s t-test (two-tailed), **p < 0.01 (p = 0.0016). (I) Quantitative data of cell population. Results are presented as the mean ± SEM (Control: n = 6, LHON: n = 4).

Figure 3

mtDNA-related genes and mitochondrial activity in LHON-RO. (A–C) The expression levels of mRNA for MT-ND4, MT-ATP6, and MT-COX1 were measured by qPCR of ROs at Day 35. Mean ± SEM (Control: n = 12, LHON: n = 8). Student’s t-test (two-tailed), *p < 0.05 (B: p = 0.046) **p < 0.01 (A: p = 0.005). (D) Protocol for MT1 assay. (E) Representative images of Day-37 ROs stained with MT1 (magenta). (F) The intensity of MT1 expression was measured. Scale bar = 50 μm. Mean ± SEM (Control: n = 12, LHON: n = 8). Student’s t-test (two-tailed), **p < 0.01 (p = 0.003). (G) Quantification of ATP production in Day-35 ROs. Mean ± SEM (Control: n = 17, LHON: n = 16). Student’s t-test (two-tailed), ***p < 0.001.

Figure 4

Idebenone increased the RGC number in LHON-RO. (A) Protocol for idebenone treatment. (B) Representative images of ROs treated with idebenone (2 or 10 μM) for 7 days, immunostained with BRN3 (magenta). (C) The number of RGCs in the entire RO was counted. Scale bar = 100 μm. Mean ± SEM (n = 4, 5). Dunnett’s test, **p < 0.01 (p = 0.004). (D) Quantification of ATP production in Day 42 ROs treated with idebenone (10 μM) for 7 days. Mean ± SEM (n = 8, 9). Student’s t-test (two-tailed), **p < 0.01 (p = 0.008).

Figure 5

Mitophagy was reduced in the LHON-RO model. (A) Extraction of enriched genes in LHON-RO not LHON-FB, as shown in the Venn diagram. Among the differentially expressed genes, overexpressed gene ontology (GO) terms were identified. (B,C) Gene set enrichment analysis of autophagy related genesets in LHON-RO. (D) Control RO and LHON RO was analyzed by Western blotting. The representative immunoblots show the expression of PINK1, Parkin, LC3-I, LC3-II, p62 and β-actin in the cell lysate of RO. (E–H) Quantitative analysis of PINK1 (E), Parkin (F), LC3-II/LC3-I (G) and p62 (H) expression. Mean ± SEM (n = 6). Student’s t-test (two-tailed), *p < 0.05, **p < 0.01 ***p < 0.001 (E: p = 0.0001, F: p = 0.0136, G: p = 0.0290, H: p = 0.0077).