Mitochondrial replacement in an iPSC model of Leber's hereditary optic neuropathy

Abstract

Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

Keywords: Leber’s hereditary optic neuropathy; cybrid; disease model; induced pluripotent stem cells; retinal ganglion cells.

Figure 1. Using cybrid transfer to generate mutation-free LHON fibroblasts and iPSCs

(A) Diagram of cybrid generation. Fibroblasts were pre-treated with the mitochondrial toxin rhodamine 6-G (R6G) then fused with healthy donor mitochondria obtained from normal keratinocytes. On day 29-32, proliferating colonies were picked and expanded.

Figure 2. LHON disease phenotype in iPSC-derived RGCs can be reversed in corrected cybrid lines

(A) Efficiency in RGC differentiation as assessed by the % of THY1.1 positive cells obtained post MACS sorting for control, LHON and corrected cybrid lines. Data are expressed as mean + SEM of independent samples (n=11 for control, n=8 for LHON and n=8 for corrected cybrid, expressed as pooled data of experimental repeats and biological repeats (3 clones)) (B) TUNEL assay revealed increased susceptibility to apoptosis in LHON RGCs and reversal in corrected cybrid lines. Data are expressed as mean of each clone, n = 3 clones, error bars = mean ± SEM. (C) Quantification of mitochondrial superoxide levels using MitoSOX in control, LHON and corrected cybrid RGCs. Error bars = ± SEM, n = 3 clones. Statistical significance was established by one way ANOVA followed by Dunnett's test for multiple comparisons, ** p<0.01, * p<0.05, ns: not significant.