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Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

ABSTRACT: Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in mitochondrial functions, metabolic pathways, and extracellular cellular matrix reorganisation. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL, including proteins involve in mitochondrial biology and neuodegeneration. Investigation of mitochondrial functions in the two cohorts confirmed a modification of respiration etc…. This study provides strong proof of concept of the validity of using iPSC for the modeling of complex diseases. It is the first to use a large scale patient -derived iPSC cohort to uncover important differences in RPE homeostasis associated with geographic atrophy. It clearly identifies mitochondrial activity as a core constitutive difference of the RPE from patients with geographic atrophy, and could be a target of potential therapies for this condition (STACEY/MATT).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

DISEASE(S): Age Related Macular Degeneration

SUBMITTER: Mehdi Mirzaei

LAB HEAD: Prof. Alice Pebay

PROVIDER: PXD029501 | Pride | 2022-06-01

REPOSITORIES: Pride

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			Action	DRS
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	480401_ea04435_Meh_hrMS2.mzIdentML	Mzid
	480402_ea04436_Meh_hrMS2.mzIdentML	Mzid
	480403_ea04437_Meh_hrMS2.mzIdentML	Mzid
	480404_ea04439_Meh_hrMS2.mzIdentML	Mzid

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Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. Secondary MEFs were isolated from E13.5 embryos, which were harvested by tetraploid complementation. Secondary MEFs were plated on gelatin-coated 6-well plates at a density of 1x105 cells/well in the complete ES medium. After 24 h incubation, secondary MEFs were fed with complete ES medium with or without Dox (1.5 ?g/ml) and with or without 200 nM Tmx. Culture medium was changed every day. Withdrawal of drugs (Dox or Tmx) was always followed by 1x washing by PBS before changing culture medium. We cotransfected MEFs with PB-Tet-MKOS and either a PB-Tet-Zscan4 or PB-Tet-DsRed (control). These cells were subsequently cultured in the standard iPSC generation condition (Dox+). Unexpectedly, we found that MEFs transfected with a PB-Tet-Zscan4 show consistently higher efficiency of iPSC generation than MEFs transfected with a PB-Tet-DsRed (control). All selected iPSCs expressed pluripotent marker genes according to RT-PCR analysis and were positive for ALP, Nanog, and SSEA-1. These iPSCs also formed embryoid bodies in hanging drops without LIF and could be differentiated in vitro to cells of all three germ layers. We are currently studying whether the presence of Zscan4 has the beneficial effects on iPSCs in terms of genome stability.

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Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

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