Study Design

We have utilized an existing repository of AMD patient clinical data (AREDS2) that includes 5-10 years of extensive clinical imaging and medical history, combined with genomic data to generate a comprehensive set of iPSC-derived RPE for disease pathway analysis and genotype-phenotype association. In vitro analysis of iPSC-RPE can be used to link RPE signaling pathways to AMD risk alleles and thus to various stages of AMD. This allows making a direct link between RPE signaling pathways, AMD risk alleles, and AMD phenotype. To help accomplish this goal we have established a collaboration with the New York Stem Cell Foundation (NYSCF) who have generated over 60 AMD iPSC-derived RPE and, in some few cases, twin controls and provide storage and distribution of these cell lines to the entire scientific community.

Specific Example

Recent literature (Pandey et al, 2017 Nature; Calippe et al., 2017 Immunity) links non-canonical activation of complement (specifically C5a and C3a – complement anaphylatoxins) to AMD initiation. Recently, in vitro activation of non-canonical complement in RPE cultures has been linked to changes in RPE structure, increased “drusen” formation, reduced autophagy, increased activation of inflammasome and inflammatory cytokines, and increased activation of NFƙb signaling pathway (through CD88 and TLR4 receptors) (Pilgrim et al., 2017 IOVS; (Celkova, et al., 2015 J. Clin. Med.). Downstream activation of miR-155, under circadian control, as well as RPE physiology changes have been determined (NEI IRP data). Based on these findings, high-throughput assays will be performed on iPSC-RPE. To mimic the initiation of AMD pathogenesis in vitro we will treat iPSC-RPE monolayers with C5a/C3a competent human serum and quantitatively analyze:

RPE characteristics

RPE shape, pigmentation, and autofluorescent changes using live-imaging (software developed at NEI IRP). These findings will be correlated to in vivo changes in RPE shape and pigmentation and analyzed longitudinally in patients using adaptive optics.

Drusen-like particles

Formation of drusen-like particles and their polarized secretion by iPSC-derived RPE cells (Pilgrim et al., 2017, IOVS) to determine a direct relationship between AMD genetics and drusen or pseudoreticular drusen formation.


Specific intracellular autophagic pathways using fluorescent reporters (Barmada et al., 2014) to identify potentially druggable targets to slow down disease initiation or progression (The HD iPSC Consortium, Nat. Neuroscience, epub March 2017 – Analysis of iPSC-neurons derived from Huntington disease patients demonstrates that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. Some of these phenotypes could be reversed by the treatment of isoxazole-9).


The polarized secretion by human RPE inflammatory cytokines - IL6, Il1-beta, IL18, and VEGF as well as the expression of miRNAs (eg miRs-155, 204) that have recently been shown to activate RPE inflammasome activity and AMD pathophysiology (Shi et al., 2008, IOVS; Wang et al., 2010 Hum. Gen.; Ambati et al., 2013 Nat. Rev. Immunology; Campbell, 2014 Adv. Exp. Med. Biol; Celkova, et al., 2015 J. Clin. Med; Berber et al., 2017 Mol. Diagn. Ther.).