Investigating retinal pathology in patients carrying m.3243A>G mutation using human induced pluripotent stem cells

Abstract

The heteroplasmic mutation in the mitochondrial gene MT-TL1 encoding tRNALeucine (UUR) at nucleotide position 3243 resulting in the arginine to guanine transition (m.3243A>G) is the most common pathogenic mutation in the mitochondrial genome. Originally associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome, it has also been linked to a number of other clinical phenotypes. It is common for patients harbouring the mutation to develop a range of ocular abnormalities, including those affecting retinal pigment epithelium (RPE) cells. The underlying mechanisms of RPE degeneration remain unclear. Using fibroblasts derived from patients with retinal changes with the m.3243A>G, I generated heteroplasmic human induced pluripotent stem cell (hiPSC) clones harbouring the m.3243A>G mutation. RPE cells differentiated from patient hiPSCs displayed typical cobblestone morphology and expressed mature RPE-associated markers. The RPE cells retained their ability to form blood-retinal barrier as assessed by measuring transepithelial resistance. However, cells with high levels of the m.3243A>G showed reduced propensity for pigment formation. Additionally, the RPE cells contained abnormal mitochondria and melanosomes, which is likely to manifest as a reduced ability to absorb stray light. These findings have remarkable similarities to the ones seen in RPE cells described in post mortem tissues of patients with the m.3243A>G mutation. In addition, patient cells showed defects in phagocytosis of photoreceptor outer segments, a functional defect associated with other retinal diseases. Overall, the results provide an indication that RPE cells with the m.3243A>G have reduced ability to perform at least two of their main functions: absorption of stray light and phagocytosis, suggesting possible pathological processes associated with ocular symptoms seen in patients. The ability to mimic these manifestations in vitro would allow investigating pathological mechanisms further and allow testing novel therapeutic agents aimed at alleviating or treating the symptoms.