Furthermore, we demonstrate the ability of our RPE monolayers to recapitulate key features of AMD, including drusen‐like deposits, when exposed to chronic oxidative stress, and use this system to analyse the dynamics of EV release and protein cargo in homeostatic and AMD‐like environments. Here, we demonstrate that the RPE tissue present in our hiPSC‐derived retinal organoids is analogous to the native human RPE and establish a method to derive functionally mature polarised RPE monolayers analogous to human primary RPE. Human induced pluripotent stem cells (hiPSCs) (Takahashi & Yamanaka, 2006) provide unprecedented opportunities for the development of human cell‐based models to study diseases (Canto‐Soler et al., 2016). Notably, to date, the characterisation of the proteome cargo of EVs from human RPE tissue in homeostatic conditions, and analysis of changes induced by an AMD‐like environment have not been accomplished. Thus, the cargo contained in RPE‐derived EVs during AMD may reflect the type and physiological–pathological state of the RPE cells. It has been observed that EV cargo reflects the nature and physiology of their cell of origin, and any change in cell homeostasis might modify the molecular composition of EVs (Baixauli et al., 2014 Colombo et al., 2014). Importantly, while the mechanisms of propagation of RPE dysfunction in AMD remain a critical gap in our understanding of the disease, EVs play a role in spreading the toxic forms of aggregated proteins in other neurodegenerative diseases (Alvarez‐Erviti et al., 2011 Rajendran et al., 2006). EVs have been defined as a double‐edged sword since they can both, promote disease progression or support homeostasis maintenance (Baixauli et al., 2014 Xu et al., 2018). A better understanding of this process could lead to therapeutic strategies to modulate drusen biogenesis, and in turn, to slow or halt the progression of AMD.Įmerging evidence suggests that extracellular vesicles (EVs), such as exosomes, microvesicles and exomeres, may participate in the pathogenesis of AMD (Klingeborn et al., 2017 Lakkaraju et al., 2020).
Although drusen are widely accepted as contributors to the etiology of both dry and wet AMD, little is known about its biogenesis. Advanced dry AMD is characterised by focal atrophy of the RPE and loss of macular photoreceptors, whereas choroidal neovascularisation, also known as wet AMD, involves abnormal blood vessel growth from the choriocapillaris through the RPE (Bhutto & Lutty, 2012). The presence of numerous drusen in the macula is considered a major risk factor for the development of advanced age‐related macular degeneration (AMD) (Ambati et al., 2003), a leading cause of blindness and visual impairment, affecting millions of individuals worldwide (Wong et al., 2014). Collectively, our results strongly support an active role of RPE‐derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.Ī hallmark of ageing in the eye is the appearance of drusen, extracellular deposits that form between the basal lamina of the RPE and the inner collagenous layer of the Bruch's membrane (Green, 1999). These observations underpin the existence of a finely‐tuned mechanism regulating directional apical:basal sorting and secretion of drusen‐associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Notably, drusen‐associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. Furthermore, we provide first evidence that drusen‐associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. Age‐related macular degeneration (AMD) is a leading cause of blindness worldwide.
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