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

PhD ThesisThe 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.Dr William Edmund Harker Foundation, ERC (#614620) and Northern Academic Health Sciences Network for funding this project

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent

Induced pluripotent stem cell (iPSC)‐derived retinal organoids provide a platform to study human retinogenesis, disease modeling, and compound screening. Although retinal organoids may represent tissue structures with greater physiological relevance to the in vivo human retina, their generation is not without limitations. Various protocols have been developed to enable development of organoids with all major retinal cell types; however, variability across iPSC lines is often reported. Modulating signaling pathways important for eye formation, such as those involving bone morphogenetic protein 4 (BMP4) and insulin‐like growth factor 1 (IGF1), is a common approach used for the generation of retinal tissue in vitro. We used three human iPSC lines to generate retinal organoids by activating either BMP4 or IGF1 signaling and assessed differentiation efficiency by monitoring morphological changes, gene and protein expression, and function. Our results showed that the ability of iPSC to give rise to retinal organoids in response to IGF1 and BMP4 activation was line‐ and method‐dependent. This demonstrates that careful consideration is needed when choosing a differentiation approach, which would also depend on overall project aims

Room temperature shipment does not affect the biological activity of pluripotent stem cell-derived retinal organoids

The generation of laminated and light responsive retinal organoids from induced pluripotent stem cells (iPSCs) provides a powerful tool for the study of retinal diseases and drug discovery and a robust platform for cell-based therapies. The aim of this study is to investigate whether retinal organoids can retain their morphological and functional characteristics upon storage at room temperature (RT) conditions and shipment by air using a commercially available container that maintains the environment at ambient temperature. Morphological analysis and measurements of neuroepithelial thickness revealed no differences between control, RT incubated and shipped organoids. Similarly immunohistochemical analysis showed no differences in cell type composition and position within the laminated retinal structure. All groups showed a similar response to light, suggesting that the biological function of retinal organoids was not affected by RT storage or shipment. These findings provide an advance in transport of ready-made retinal organoids, increasing their availability to many research and pharma labs worldwide and facilitating cross-collaborative research

Human iPSC disease modelling reveals functional and structural defects in retinal pigment epithelial cells harbouring the m.3243A > G mitochondrial DNA mutation.

The m.3243A > G mitochondrial DNA mutation was originally described in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The phenotypic spectrum of the m.3243A > G mutation has since expanded to include a spectrum of neuromuscular and ocular manifestations, including reduced vision with retinal degeneration, the underlying mechanism of which remains unclear. We used dermal fibroblasts, from patients with retinal pathology secondary to the m.3243A > G mutation to generate heteroplasmic induced pluripotent stem cell (hiPSC) clones. RPE cells differentiated from these hiPSCs contained morphologically abnormal mitochondria and melanosomes, and exhibited marked functional defects in phagocytosis of photoreceptor outer segments. These findings have striking similarities to the pathological abnormalities reported in RPE cells studied from post-mortem tissues of affected m.3243A > G mutation carriers. Overall, our results indicate that RPE cells carrying the m.3243A > G mutation have a reduced ability to perform the critical physiological function of phagocytosis. Aberrant melanosomal morphology may potentially have consequences on the ability of the cells to perform another important protective function, namely absorption of stray light. Our in vitro cell model could prove a powerful tool to further dissect the complex pathophysiological mechanisms that underlie the tissue specificity of the m.3243A > G mutation, and importantly, allow the future testing of novel therapeutic agents

An iPSC Patient Specific Model of CFH (Y402H) Polymorphism Displays Characteristic Features of AMD and Indicates a Beneficial Role for UV Light Exposure

Age related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease, however these do not exist for the dry form. Complement factor H (CFH) polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, retinal pigment epithelium (RPE) damage and visual decline. We have derived and characterised induced pluripotent stem cell (iPSCs) lines from two patients without AMD and low risk genotype and two patients with advanced AMD and high risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H (FH), factor I (FI) and factor H like 1 (FHL-1). The iPSC RPE cells derived from high risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy and deposition of “drüsen” like deposits. The low and high risk RPE cells respond differently to intermittent exposure to UV light which leads to an improvement in cellular and functional phenotype only in the high risk AMD-RPE cells. Taken together our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing