Centre for Eye Research Australia (CERA) - Research Publications

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Author: Guymer, Robyn × Author: Hewitt, Alexander × Start date: 2020 ×

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    A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells
    Hall, J ; Daniszewski, M ; Cheung, S ; Shobhana, K ; Kumar, H ; Liang, HH ; Beetham, H ; Cho, E ; Abbott, C ; Hewitt, AW ; Simpson, KJ ; Guymer, RH ; Paull, D ; Pebay, A ; Lidgerwood, GE (Elsevier, 2024-06)
    Age-Related Macular Degeneration (AMD) is a highly prevalent form of retinal disease amongst Western communities over 50 years of age. A hallmark of AMD pathogenesis is the accumulation of drusen underneath the retinal pigment epithelium (RPE), a biological process also observable in vitro. The accumulation of drusen has been shown to predict the progression to advanced AMD, making accurate characterisation of drusen in vitro models valuable in disease modelling and drug development. More recently, deposits above the RPE in the subretinal space, called reticular pseudodrusen (RPD) have been recognized as a sub-phenotype of AMD. While in vitro imaging techniques allow for the immunostaining of drusen-like deposits, quantification of these deposits often requires slow, low throughput manual counting of images. This further lends itself to issues including sampling biases, while ignoring critical data parameters including volume and precise localization. To overcome these issues, we developed a semi-automated pipeline for quantifying the presence of drusen-like deposits in vitro, using RPE cultures derived from patient-specific induced pluripotent stem cells (iPSCs). Using high-throughput confocal microscopy, together with three-dimensional reconstruction, we developed an imaging and analysis pipeline that quantifies the number of drusen-like deposits, and accurately and reproducibly provides the location and composition of these deposits. Extending its utility, this pipeline can determine whether the drusen-like deposits locate to the apical or basal surface of RPE cells. Here, we validate the utility of this pipeline in the quantification of drusen-like deposits in six iPSCs lines derived from patients with AMD, following their differentiation into RPE cells. This pipeline provides a valuable tool for the in vitro modelling of AMD and other retinal disease, and is amenable to mid and high throughput screenings.
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    Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration
    Senabouth, A ; Daniszewski, M ; Lidgerwood, GE ; Liang, HH ; Hernandez, D ; Mirzaei, M ; Keenan, SN ; Zhang, R ; Han, X ; Neavin, D ; Rooney, L ; Sanchez, MIGL ; Gulluyan, L ; Paulo, JA ; Clarke, L ; Kearns, LS ; Gnanasambandapillai, V ; Chan, C-L ; Nguyen, U ; Steinmann, AM ; McCloy, RA ; Farbehi, N ; Gupta, VK ; Mackey, DA ; Bylsma, G ; Verma, N ; MacGregor, S ; Watt, MJ ; Guymer, RH ; Powell, JE ; Hewitt, AW ; Pebay, A (NATURE PORTFOLIO, 2022-07-26)
    There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.