Cone photoreceptors are specialized cells within the eye responsible for sensing light and starting the process of seeing. Using adaptive optics, a tool borrowed from astronomy, we can image these cells in a living human eye. Being able to track these individual cells over time in different images of the retina and from different imaging modalities is helpful for diagnosing, understanding, and finding ways to treat retinal diseases. But it is time consuming for even an expert to match cells between images from the same instrument and even more challenging between different modalities. And solving this problem is not as simple as performing an image match, because missing cells, image warping due to eye movement, and different lighting present challenges. This research investigates ways to algorithmically match cells between two images of the retina while overcoming these obstacles. We started with a basic algorithm to match an image of cells to itself, and then we made iterative improvements and additions to the algorithm as we ran it on images with more differences in warping and missing cells, improving its robustness. The result was a specialized algorithm to match cells based only on the local patterns of neighboring cells, which solved the problem of global warping and different lighting because local cell locations are unaffected by these factors. This algorithm helps automate the process of matching cells across multiple images taken at different times and with different instruments, and decreases the time to do so from minutes or hours to just seconds. In the future, we hope to make this algorithm more resilient to missing cells by interpolating matches in regions between areas where we have confident matches. By automating this matching task, we can aid and speed up research into these cells that are progressively affected by retinal diseases.

Age-related macular degeneration (AMD) is the leading cause of vision loss for people aged 50 years and older. Variants in the complement factor H (CFH) gene are associated with an increased risk of developing AMD, making it one of the main drivers for disease progression. Rare mutations that affect the expression of CFH protein and its isoform - Factor H-like protein 1 (FHL-1), have been linked to early-onset macular drusen (EOMD), an inherited degenerative disease with similar clinical characteristics to AMD. Our lab recently found a novel CFH variant of two family members with EOMD. This single nucleotide polymorphism (SNP) in the conserved splice site of intron 3 resulted in a frameshift with premature stop codon and no translation, causing ~50%reduction in CFH/FHL-1 production. Since CFH/FHL-1 are cofactors for C3b cleavage in the alternative pathway of the complement system, this haploinsufficiency will likely affect complement regulation in retinal pigmented epithelial (RPE) cells, hence contributing to the development of AMD-like pathology. Using patient specific iPSC-derived RPE cells, this study aims to investigate the role of CFH/FHL-1 in maintaining complement homeostasis. I will examine the expression levels of several active complement components and multiple regulators of complement activation (RCA). ELISA and Western blots will be used to quantify protein expression, and protein localization was revealed through immunostaining. RPE cells will also be exposed to normal human serum and stained for the membrane attack complex (MAC) to assess the susceptibility of cells to complement-mediated damage. I will also examine reversal of phenotype in EOMD RPE supplemented with exogenous CFH or FHL-1, as well as in Crispr-corrected EOMD iPSC-RPE cells. Results from this study will provide insights into the role CFH/FHL-1 plays in regulating players of the complement pathway RPE cells and its contribution towards progression of AMD.

Age-related macular degeneration (AMD) is a multifactorial eye disease characterized by drusen formation and thickening of the Bruch’s membrane (BrM). The BrM is a extracellular matrix (ECM) located between the retinal pigment epithelium (RPE) and choriocapillaris, which play roles in structural support, transport of nutrients and interactions with RPE. Changes to the ECM composition and architecture, contributed partly by accumulation of basal deposits during AMD could interfere with nutrient diffusion and transport. Moreover, RPE cells perceive changes that occur in the ECM, therefore age-related changes that occur in the Bruch’s membrane can affect RPE pathology in AMD cells. Early onset macular drusen (EOMD) is an inherited retinal degeneration with similar clinical phenotype to AMD. Patients with EOMD have rare genetic variants in the complement factor H (CFH) gene that affects both CFH and factor H-like protein 1 (FHL-1) expression. We recently described a novel mutation in a EOMD family, which resulted in CFH and FHL-1 haploinsufficiency. Peripheral blood mononuclear cell were isolated from EOMD patients, reprogrammed into induced pluripotent stem cells (iPSC), and differentiated into RPE cells. Preliminary results indicated the cells to have phenotypes consistent with early AMD, making it a beneficial in vitro model for the study of the disease. The ECM composition has not yet been explored in the EOMD RPE cells, therefore the purpose of this project is to examine the expression of and staining pattern of ECM components, as well as several common drusen components. RPE seeded on transwell filters were collected, sectioned and immunostaining was performed to visualize the ECM components. Expression was further validated by western blot. Results from this study will determine if CFH and FHL-1 haploinsuffiency leads to alteration of ECM, which could play a contributing role towards pathology of the RPE.