Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut.
The New York Stem Cell Foundation (NYSCF) Research Institute, New York, New York.
Stem Cells Transl Med. 2020 Mar;9(3):364-376. doi: 10.1002/sctm.19-0321. Epub 2019 Dec 16.
Modeling age-related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch's membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch's membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age-matched (normal) controls. RPE derived from iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell attachment and viability was tested on nitrite-modified ECM, a typical modification of aged Bruch's membrane. DNA microarrays with hierarchical clustering and analysis of mitochondrial function were used to elucidate possible mechanisms for the observed phenotypes. Differentiated RPE displayed cell-specific morphology and markers. The TER and phagocytic capacity were similar among iPSC-derived RPE cultures. However, distinct clusters were found for the transcriptomes of AMD and control iPSC-derived RPE. AMD-derived iPSC-RPE downregulated genes responsible for metabolic-related pathways and cell attachment. AMD-derived iPSC-RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite-modified ECM. Cells that did attach induced the expression of complement genes. Despite reprogramming, iPSC derived from AMD patients yielded RPE with a transcriptome that is distinct from that of age-matched controls. When challenged with an AMD-like modification of Bruch's membrane, AMD-derived iPSC-RPE activated the complement immune system.
模拟年龄相关性黄斑变性（AMD）具有挑战性，因为它是一种多因素疾病。为了专注于视网膜色素上皮（RPE）和布鲁赫膜之间的相互作用，我们从 AMD 患者中生成了 RPE，并使用了模拟老年布鲁赫膜的改变细胞外基质（ECM）。诱导多能干细胞（iPSC）是从 AMD 患者或年龄匹配的（正常）对照的成纤维细胞中产生的。通过形态学、标志物表达、跨上皮电阻（TER）和杆状光感受器外节的吞噬作用来分析 iPSC 衍生的 RPE。在亚硝酸盐修饰的 ECM 上测试细胞附着和活力，这是老年布鲁赫膜的典型修饰。使用层次聚类和线粒体功能分析的 DNA 微阵列来阐明观察到的表型的可能机制。分化的 RPE 显示出细胞特异性的形态和标志物。iPSC 衍生的 RPE 培养物之间的 TER 和吞噬能力相似。然而，AMD 和对照 iPSC 衍生的 RPE 的转录组发现了不同的聚类。AMD 衍生的 iPSC-RPE 下调了与代谢相关途径和细胞附着有关的基因。AMD 衍生的 iPSC-RPE 表现出减少的线粒体呼吸和附着在亚硝酸盐修饰的 ECM 上并存活的能力。附着的细胞诱导补体基因的表达。尽管重编程，来自 AMD 患者的 iPSC 产生的 RPE 的转录组与年龄匹配的对照不同。当受到类似于 AMD 的布鲁赫膜修饰的挑战时，AMD 衍生的 iPSC-RPE 激活了补体免疫系统。
