Three-dimensional retinal organoids from induced pluripotent stem cells derived from rd16 mouse model of CEP290-LCA

Abstract

Purpose : Three-dimensional retinal organoids offers a valuable platform to investigate the pathogenesis of retinal degenerative diseases and evaluate novel therapies. The goal of this study is to develop and characterize 3-D retinal organoids from induced pluripotent stem cells (iPSCs) of Nrl-GFP rd16 mouse, a model of Leber congenital amaurosis caused by CEP290 mutations, in order to identify small molecule drug candidates for treatment.

Methods : We differentiated two clones of Nrl-GFP wild type (WT) and two clones of rd16 iPSCs into retinal organoids and flow-sorted GFP+ rod photoreceptors at different stages (differentiation day (D) 18, D22, D28 and D32). Total RNA (RNA integrity number ≥ 7.0) were obtained from purified rod cells for transcriptome analysis using the Illumina platform. Principle component analysis and hierarchical clustering were applied to compare the genes profiles between rods from WT and rd16 organoids. We performed differential expression analysis (false discovery rate < 1%; fold change ≧ 2) and gene ontology analysis to identify signaling pathways associated with rd16 disease. We are also evaluating small molecules to rescue rd16 phenotypes.

Results : The two largest principle components (PC1 and PC2) were attributed to gene expression differences between WT and rd16 rods and the developmental time, respectively. The distinct separation of WT and rd16 data in PC1 and their co-projection in PC2 suggest that Cep290 mutation in rd16 leads to divergence in gene profiles but does not affect organoid maturity. Gene ontology analysis using Kegg pathway database revealed altered expression of genes related to cell-environment interactions, PI3K-Akt signaling pathway and immune response. We have also compared the transcriptome of rods differentiating in rd16 organoids with published rod transcriptome data. The rd16 organoids exhibit loss of Nrl-GFP rods and shorter survival time compared to WT organoids and we are using these phenotypes to identify novel candidates for treatment of retinal degeneration.

Conclusions : Our studies are focusing on molecular staging of retinal organoids based on in vivo transcriptome data and examining distinctions between in vitro and in vivo disease modeling. Current ongoing work includes validation of high-throughput screening of retinal organoids to identify drug candidates for alleviating retinal degenerative diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.