Abstract
BackgroundStudy of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms.MethodsGeneration of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping.ResultsThe AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE as compared to normal RPE-iPSC-RPE.ConclusionsOur studies suggest SIRT1/PGC-1α as underlying pathways contributing to AMD pathophysiology, and open new avenues for development of targeted drugs for treatment of this devastating neurodegenerative disease of the visual system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1101-8) contains supplementary material, which is available to authorized users.
Highlights
Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease
While primary retinal pigment epithelium (RPE) could be used to study the disease phenotypes in AMD, they can quickly become depleted due to passaging and undergo senescence, whereas, induced pluripotent stem cells (iPSCs) can serve as an inexhaustible source that could continuously be differentiated to the RPE for maintenance of the disease model
We purified and cultured the RPE isolated from the macular region of the human eyes according to the established protocol [38] and performed the genetic study of the single nucleotide polymorphisms (SNPs) for the known AMD susceptibility loci
Summary
Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. The non-exudative or dry form of AMD is diagnosed by polymorphic deposits, called drusen, that accumulate underneath the RPE and can result in overlying macular atrophy and pigmentation [6]. Because of its complex etiology, understanding the molecular mechanisms of AMD has been challenging by lack of the appropriate in vitro model that could sufficiently recapitulate the characteristics of the disease. The available animal models fall short in accurately representing the characteristics of AMD due to absence of human genetic polymorphisms and long-term exposure to oxidative stress and environmental factors [8]
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