Biology subjects

Wiley, L. A.

Publications and source records attributed to Wiley, L. A..

5 recordsLinked to original sources

bioRxiv2026

Transcriptomic Characterization of Terminal Complement Complex-bound Cells in Human Choroid Using Single-Cell RNA Sequencing

Age-related macular degeneration (AMD) is among the leading causes of blindness worldwide. Early AMD is characterized by dysfunction in the choroid, including early dropout of endothelial cells and increased deposition of the complement cascade's membrane attack complex (MAC) in the choriocapillaris. In this study, we used a single-cell RNA sequencing-based approach with barcoded antibodies to measure abundance of the MAC and other surface proteins at single cell resolution on RPE/choroid samples from four aged human donor eyes. We included antibodies to detect the MAC, CD34, CD45, and complement regulators CD55 and CD59, in addition to control antibodies. Our analysis of these data revealed cell clusters with expected gene expression profiles and antibody-based detection of CD34 and CD45 congruent with transcriptome-based cell identity. We also detected surface complement regulators CD55 and CD59 across a wide variety of cell types. Across endothelial cells, surface CD55 and CD59 appeared more abundant on venous clusters, and abundance of each was correlated with expression of a third complement regulator, clusterin (CLU). The MAC was detected on a variety of cell types, but was most abundant on the surface of cells in the macrophage family, smooth muscle cells, and pericytes. We confirmed these findings by identifying MAC deposition on choriocapillaris pericytes using immunohistochemistry for MAC, endothelial, and pericyte markers. Ultimately, these data showcase a valuable new approach to analyze gene expression and surface complement in human donor eyes, and provide novel insight into patterns of MAC deposition and complement protection in the aging human choroid.

cell biology↗

bioRxiv2026

Metabolic Analysis of Human Retinal Pigment Epithelium and Choroid Tissue in Aging and Macular Degeneration

Age-related macular degeneration is a common ocular disease that causes vision loss in the elderly, with a complex set of risk factors and proposed mechanisms of pathogenesis. A powerful method for investigating changes in disease is metabolomics, by which small molecules can be identified and quantified simultaneously. We report here the metabolic analysis of human RPE-choroid tissue in aging and macular degeneration (AMD), as well as comparisons of human macular and extramacular RPE-choroid and neural retina. Levels of 215 metabolites were determined in young donors, AMD donors (early/intermediate, geographic atrophy, and neovascularization) and age-matched controls. The largest number of metabolite differences were observed between young and healthy aged controls, as opposed to between aged controls and any stage of AMD. Two notable metabolites found to be increased in aging choroids are trimethylamine N-oxide and uric acid, both of which were significant after Bonferroni correction. A mouse endothelial cell line treated with a high concentration of uric acid exhibited reduced migration in a wound closure assay. This study provides initial insights into the metabolome of human choroids in varying states of age and macular degeneration, as well as functional implications of these changes in the aging choroid.

cell biology↗

bioRxiv2025

Modeling MEK-inhibitor Associated Retinopathy in vitro using human induced-1 pluripotent stem cell-derived retinal pigment epithelial cells

Pharmacologic inhibitors of MEK are important anti-cancer drugs but can result in MEK inhibitor-Associated Retinopathy (MEKAR) in which vision is lost due to serous retinal detachments that form via an unknown mechanism. We hypothesized that the cause of this side effect is drug-induced dysfunction of retinal pigment epithelial (RPE) cells. To test this hypothesis, we used human induced pluripotent stem cell-derived RPE cells. We treated mature, hiPSC-derived RPE cells with selumetinib and measured impacts on RPE-specific function, structure, and gene expression. Selumetinib increases the ability of hiPSC-derived RPE to internalize bovine rod outer segments (1.9 vs 3.0, p=0.0024). It also decreases expression of aquaporin 1 during the first 10 days of treatment (2.7 vs 1.1, p=0.0015). It has no effect on the ability of hiPSC-derived RPE to maintain membrane integrity. Selumetinib alters gene expression of hiPSC-derived RPE, with significant changes in genes involved in transport of ions and small molecules regulating cell volume and lysosomal acidification. Selumetinib may lead to subretinal fluid accumulation by both increasing secretions into this space and decreasing outflow.

molecular biology↗

bioRxiv2025

Sialoglycoconjugate Profiling of Human Choroid, Retinal Pigment Epithelium, and Macular Degeneration Related Lesions

Age-related macular degeneration is a leading cause of central vision loss in the elderly. Early hallmarks of the disease include basal laminar deposit and choriocapillaris degeneration. The location and composition of sialoglycoconjugates in healthy and diseased choroid and disease-related lesions have not been thoroughly examined. This study utilized lectins to examine sialoglycoconjugates in human tissue, specifically Sambucus nigra/Elderberry Bark Lectin (EBL) and Maackia amurensis lectin II (MAL-II), to examine -2,6 and -2,3 sialic acids, respectively. EBL and MAL-II both label the choroid and basal laminar deposit, with slightly different patterns. Whereas MAL-II predominantly labels the choriocapillaris endothelium, EBL also labels Bruchs membrane and extracellular domains surrounding the vasculature (intercapillary pillars). EBL labeling overlaps with the distribution of complement factor H to a greater extent than MAL-II. After treatment with neuraminidase to remove terminal sialic acids, a battery of lectins was applied to sections of choroids. Lectins that recognize {beta}-galactose, N-acetyllactosamine, galactose ({beta}-1,3) N-acetylgalactosamine, and - or {beta}-N-acetylgalactosamine showed increased reactivity, including increased labeling of glycans in basal laminar deposits. This study provides insight into the location and partial identities of sialoglycoconjugates in the human choroid, with possible implications for the pathogenesis of macular degeneration.

cell biology↗

bioRxiv2025

Production of clinical grade patient iPSC-derived 3D retinal organoids containing transplantable photoreceptor cells

Neurodegenerative conditions that affect the retina are currently the leading cause of incurable blindness in the developed world. Although gene and drug therapies are being developed to slow disease progression in some cases, restorative cell replacement approaches are needed for patients with significant vision impairment due to retinal degeneration. While a variety of different cell types have been evaluated in the context of retinal cell replacement, induced pluripotent stem cells (iPSCs), which can be generated and delivered as an autologous therapeutic, are in many ways the most attractive donor cell source currently available. Like embryonic stem cells, iPSCs must be differentiated into the target therapeutic cell type prior to transplantation. For instance, for patients with retinitis pigmentosa who have primary photoreceptor cell disease, photoreceptor cell derivation and enrichment are required prior to transplantation. Although other effective retinal differentiation protocols exist, they are often not fully compatible with clinical manufacturing. In this study, we report development of a xeno-free 3D retinal differentiation protocol based on the most robust adherent/non-adherent 3D differentiation strategies published to date. In addition, we demonstrate that while iPSC reprogramming efficiency is enhanced under reduced oxygen tension (i.e., 5%), efficient embryoid body and subsequent retinal organoid production require standard oxygen levels (i.e., 21%). Finally, we show that photoreceptor precursor cells obtained from 3D retinal organoids derived using the developed protocol under current good manufacturing practices (cGMP) survive in the subretinal space of dystrophic Pde6b-null rats for 1-month post-transplantation and form new synaptic connections with host bipolar neurons.

neuroscience↗