Retinal toxicity is an adverse side effect that can cause (usually irreversible) deterioration of a person’s vision, when taking certain medication. This has been seen for example in people taking Hydroxychloroquine for Malaria due to toxicity to the retinal cells.
The retinal pigmented epithelium (RPE) is a single layer of pigmented cells that face the neurosensory retina. The RPE forms one of the blood-retina barriers of the eye and is vital for maintaining homeostasis of the retina (Straub, 2016). Disruption of one or more functions of the RPE can occur by mutation, environmental or pharmacologic interference, and lead to a range of disease states or toxic responses (Andres et al. 2015, Mecklenburget et al. 2007).
The retinal toxicity of certain medications can lead to irreversible vision loss, hence accurate assessment of the drug, and its potential adverse effects, is essential. Currently, animal models are widely used to assess the toxicity of topically and systemically applied drugs. However, the structure and physiology for commonly used regulatory species (such as rats and dogs) are distinct from that seen in humans (Shafaie et al, 2016). There are protocols for culturing ex vivo human cells, but these cells are difficult to obtain and have inherent variability meaning studies can be difficult to compare (Kuznetsova et al, 2014). There is currently no established and validated in vitro model of human RPE, but the increased development of therapies targeting ocular diseases means there is a critical need.
The team at Newcells Biotech Ltd have developed an alternative in vitro model of the retinal pigmented epithelium, to the animal models. The in vitro model of the RPE is derived from healthy and diseased human induced pluripotent stem cell (iPSC) lines. This overcomes the availability issues and variability seen in primary cell culture, opening up the potential to assess safety in healthy cell models and efficacy of novel ocular compounds in diseased cells. This model also aims to assist the industry to meet the UK government targets for the development of alternatives to animal testing.
The Newcells model is composed of a monolayer of RPE cells cultured in 24-well plate transwell inserts. The RPE cells form an epithelial barrier with the Zona Occludens protein expression required for maintenance of osmotic balance. The model has demonstrated apical-basal polarity, evaluated by expression patterns of key proteins (MertK and Collagen IV), trans-epithelial electrical resistance (TEER) comparable to that seen in ex vivo human eyes (Quinn et al, 1992) and, functionality in terms of RPE cell phagocytosis of photoreceptor outer segments (Buskin et al, 2018) by flow cytometry. It is also possible to interrogate mechanisms of toxicity using single cell RNAseq to provide information on which pathways are induced by toxins. These characterisations confirm that the RPE monolayer is a close model of the in vivo human RPE.
The Newcells RPE model should be of great interest to the pharmaceutical industry as it could better predict the outcomes in the human eye as well as reducing the number of animals used in the assessment of ocular toxicity.
Figure 1. Immunohistochemistry images of control and MertK- (F119) retinal pigmented epithelia derived from iPSC cell lines showing the polarised expression of key proteins including Collagen IV, DAPI and MertK.
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Shafaie S, Hutter V, Cook MT, et al. (2016). In vitro Cell Models for Ophthalmic Drug Development Applications. BioResearch Open Access 5.1
Kuznetsova AV, Kurinov AM, and Aleksandrova MA (2014). Cell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium. Journal of Ophthalmology Article ID 801787
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Buskin et al. (2018). Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. Nature Communications 9:4234