Retinal degeneration often results in the loss of light‐sensing photoreceptors, which leads to permanent vision loss. Generating transplantable retinal photoreceptors using human somatic cell‐derived induced pluripotent stem cells (iPSCs) holds promise to treat a variety of retinal degenerative diseases by replacing the damaged or dysfunctional native photoreceptors with healthy and functional ones. Establishment of effective methods to produce retinal cells including photoreceptors in chemically defined conditions using current Good Manufacturing Practice (cGMP)‐manufactured human iPSC lines is critical for advancing cell replacement therapy to the clinic. In this study, we used a human iPSC line (NCL‐1) derived under cGMP‐compliant conditions from CD34+ cord blood cells. The cells were differentiated into retinal cells using a small molecule‐based retinal induction protocol. We show that retinal cells including photoreceptors, retinal pigmented epithelial cells and optic cup‐like retinal organoids can be generated from the NCL‐1 iPSC line. Additionally, we show that following subretinal transplantation into immunodeficient host mouse eyes, retinal cells successfully integrated into the photoreceptor layer and developed into mature photoreceptors. This study provides strong evidence that transplantable photoreceptors can be generated from a cGMP‐manufactured human iPSC line for clinical applications. Stem Cells Translational Medicine 2018;7:210–219

Citation: Garcia TY, Gutierrez M, Reynolds J, Lamba DA. Modeling the dynamic AMD-associated chronic oxidative stress changes in human ESC and iPSC-derived RPE cells. Invest Ophthalmol Vis Sci. 2015;56:7480-7488. DOI:10.1167/iovs.15-17251 PURPOSE. Here we use human embryonic stem cells (hESCs) and human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells to model chronic oxidative stress in vitro. This model allows us to understand the evolution of chronic stress response in RPE in vivo, as well as to monitor microRNAs changes. Finally, we use this in vitro model to identify a partial agonist of NRF2 that is protective against reactive oxygen species (ROS)-induced cytotoxicity.METHODS. The hESCs and hiPSCs were differentiated toward an RPE fate. Upon maturation, RPE cells were subjected to chronic oxidative stress using Paraquat (PQ). The cells were then analyzed using immunocytochemistry and quantitative RT-PCR to look for changes in gene expression and microRNA changes. Small molecules targeting NRF2 pathways were utilized to look for protection against oxidative stress-induced apoptosis.RESULTS. We show that 160 lM PQ can be used to generate a model of chronic oxidative stress in RPE cells derived from hESCs and hiPSCs. Using this model, we characterize the NRF2 pathway effectors during the early and late stages of chronic oxidative stress and identify microRNAs changes during oxidative stress. We find that hsa-miR144 modulates NRF2 activity during ROS stress. Lastly, we found a small molecule modulator of NRF2 that plays a protective role against oxidative stress-induced RPE apoptosis.CONCLUSIONS. In summary, pluripotent stem cell-derived retinal cells can be used to model retinal diseases in a dish. This can provide an unprecedented opportunity to understand the evolution of disease processes and allow us to identify novel therapeutics.Keywords: age-related macular degeneration, disease modeling, oxidative stress, microRNA, NRF2 A ge-related macular degeneration (AMD) is the leading cause of worldwide blindness in the elderly, affecting almost 15 million people in the United States. Retinal changes associated with AMD are present in approximately 10% of people over 65 years of age and as many as one in three people over the age of 80 years. Although the disease was first described in the 1800s, its etiology remains poorly understood, and multiple factors may be involved in the progression of the disorder, including chronic oxidative stress. A number of studies have associated oxidative stress as the key driver to AMD.1,2 The retina is one of the tissues with the greatest consumption of oxygen in the body. 3 This results in significant production of reactive oxygen species (ROS) in the retinal pigment epithelium (RPE). Increasing age results in the loss of ability to deal with this excessive ROS, which leads to oxidative damage.There are no known effective forms of treatment for the common dry form of AMD. This likely is due to the complex multifactorial etiology of AMD and...

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2′-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice.

Cocrystallization of Co2(CO)8 with C60 traps the cobalt carbonyl molecule as the D(3d) isomer that otherwise is not available for crystallographic analysis.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE). The molecular mechanisms leading to RPE dysfunction in response to ROS are unclear. Here, human stem cell-derived RPE samples were stressed with ROS for 1 or 3 weeks, and both intracellular and secreted proteomes were quantified by mass spectrometry. ROS increased glycolytic proteins but decreased mitochondrial complex I subunits, as well as membrane proteins required for endocytosis. RPE secreted over 1,000 proteins, many of which changed significantly due to ROS. Notably, secreted APOE is decreased 4-fold, and urotensin-II, the strongest known vasoconstrictor, doubled. Furthermore, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, our results paint a detailed molecular picture of the retinal stress response in space and time.