The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, a high prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. Ophthalmologic drug development, to date, largely relies on animal models, which often do not provide results that are translatable to human patients. Hence, the establishment of sophisticated human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs. It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.
Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.cone photoreceptor ͉ hereditary retinal disorder ͉ phosphodiesterase
Achromatopsia (ACHM) is an autosomal-recessive retinal dystrophy characterized by color blindness, photophobia, nystagmus, and severely reduced visual acuity. Its prevalence has been estimated to about 1 in 30,000 individuals. Four genes, GNAT2, PDE6C, CNGA3, and CNGB3, have been implicated in ACHM, and all encode functional components of the phototransduction cascade in cone photoreceptors. Applying a functional-candidate-gene approach that focused on screening additional genes involved in this process in a cohort of 611 index cases with ACHM or other cone photoreceptor disorders, we detected a homozygous single base change (c.35C>G) resulting in a nonsense mutation (p.Ser12(∗)) in PDE6H, encoding the inhibitory γ subunit of the cone photoreceptor cyclic guanosine monophosphate phosphodiesterase. The c.35C>G mutation was present in three individuals from two independent families with a clinical diagnosis of incomplete ACHM and preserved short-wavelength-sensitive cone function. Moreover, we show through immunohistochemical colocalization studies in mouse retina that Pde6h is evenly present in all retinal cone photoreceptors, a fact that had been under debate in the past. These findings add PDE6H to the set of genes involved in autosomal-recessive cone disorders and demonstrate the importance of the inhibitory γ subunit in cone phototransduction.
polymorphisms in the complement factor H (CFH) gene, coding for the factor H protein (fH), can increase the risk for age-related macular degeneration (AMD). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. Whether this alone suffices to trigger AMD pathogenesis remains unclear. in AMD, retinal homeostasis is compromised due to the dysfunction of retinal pigment epithelium (Rpe) cells. to investigate the impact of endogenous fH loss on Rpe cell balance, we silenced CFH in human hTERT-RPE1 cells. FH reduction led to accumulation of C3, at both RNA and protein level and increased RPE vulnerability toward oxidative stress. Mild hydrogen-peroxide exposure in combination with CFH knock-down led to a reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation, which is a key aspect of AMD pathogenesis. in parallel, cell viability was decreased. the perturbations of energy metabolism were accompanied by transcriptional deregulation of several glucose metabolism genes as well as genes modulating mitochondrial stability. our data suggest that endogenously produced fH contributes to transcriptional and metabolic homeostasis and protects Rpe cells from oxidative stress, highlighting a novel role of fH in AMD pathogenesis. Retinal pigment epithelium (RPE) cells are crucial for the maintenance of retinal homeostasis. RPE cells are located on a thin membrane called Bruch´s membrane (BM), and together provide a barrier between the neuroretina and the choroid capillary network. In addition, RPE cells fulfil several key functions, such as phagocytosis of the photoreceptor outer segments, transport of nutrients, preservation of the retinal structure and, most importantly, due to their high antioxidant capacity, RPE cells protect the retina from photo-oxidation and oxidative damage 1. RPE dysfunction and degeneration are key features of age-related macular degeneration (AMD), a complex degenerative disease, and the primary cause of blindness in the elderly population 2. AMD is characterized by a progressive degeneration of the macula, the cone-rich area of the retina, where damage in this area leads to central vision loss and ultimately blindness 3. The aetiology of AMD involves ageing processes, genetic predisposition and environmental factors, however a full understanding of AMD pathogenesis is lacking, which makes drug discovery challenging 4. A defining hallmark of AMD is the presence of deposits, called drusen, between the BM and the RPE layer 5. In the presence of drusen or altered extracellular matrix (ECM) of BM, the functionality of RPE cells may be impaired 6. The retinal microenvironment is already highly oxidized in physiological conditions, due to a very high energy demand and photo-oxidation. Ageing processes, in combination with external stressors including smoking or a high fat diet 7,8 , force RPE cells to deal with excessive levels of oxidative stress. Disturbed RPE cell homeostasis, and in particular RPE cell ...
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