Modeling the Dynamic AMD-Associated Chronic Oxidative Stress Changes in Human ESC and iPSC-Derived RPE Cells
Modeling the Dynamic AMD-Associated Chronic Oxidative Stress Changes in Human ESC and iPSC-Derived RPE Cells
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DOI：
10.1167/iovs.15-17251
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发表时间：
2015-11-01
影响因子：
4.4
通讯作者：
Lamba, Deepak A.
中科院分区：
文献类型：
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作者：
Garcia, Thelma Y.;Gutierrez, Mark;Lamba, Deepak A.
目的。在这里，我们使用人胚胎干细胞（hESC）和人诱导多能干细胞（hiPSC）衍生的视网膜色素上皮（RPE）细胞在体外模拟慢性氧化应激。该模型使我们能够了解体内 RPE 慢性应激反应的演变，并监测 microRNA 的变化。最后，我们使用这个体外模型来鉴定 NRF2 的部分激动剂，它可以防止活性氧 (ROS) 诱导的细胞毒性。 hESC 和 hiPSC 朝着 RPE 命运分化。成熟后，RPE 细胞使用百草枯 (PQ) 承受慢性氧化应激。然后使用免疫细胞化学和定量 RT-PCR 对细胞进行分析，以寻找基因表达和 microRNA 变化的变化。利用靶向 NRF2 途径的小分子来寻找针对氧化应激诱导的细胞凋亡的保护作用。结果。我们证明，160 mu M PQ 可用于在源自 hESC 和 hiPSC 的 RPE 细胞中生成慢性氧化应激模型。使用该模型，我们表征了慢性氧化应激早期和晚期的 NRF2 通路效应子，并识别了氧化应激期间 microRNA 的变化。我们发现 hsa-miR144 在 ROS 应激期间调节 NRF2 活性。最后，我们发现了一种 NRF2 小分子调节剂，它对氧化应激诱导的 RPE 凋亡具有保护作用。结论。总之，多能干细胞来源的视网膜细胞可用于在培养皿中模拟视网膜疾病。这可以提供前所未有的机会来了解疾病过程的演变，并使我们能够确定新的治疗方法。
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 mu M 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.