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: