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Donald J Zack, Alyssa Kallman, Elizabeth E. Capowski, Anu Kaushik, Liben Chen, Melissa Liu, Alex Jansen, Kim Edwards, Cindy Berlinicke, Ming-Wen Hu, Jiang Qian, Eric A Pierce, Jeff Wang, David M Gamm; Single-cell transcriptomic analysis of human NRL null retinal organoids. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4766.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative diseases remain largely untreatable, partly due to limited understanding of the complex gene-expression patterns that direct photoreceptor (PR) development. By performing single-cell transcriptomic analysis of human in vitromodels of retinal development in the presence and absence of Neural Retina-specific Leucine zipper protein (NRL), a transcription factor vital to rod PR development and degeneration, we are working to systematically define the gene networks involved in PR differentiation and degeneration.
Human induced pluripotent stem cell control and NRL null lines were differentiated into retinal organoids and samples were collected at 100 and 170 days for single cell mRNA capture and analysis using Dropseq. We analyzed the transcriptomic data by principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) to cluster cells by variably expressed genes. Cell populations were identified by marker genes and PRs were selected to reconstruct developmental trajectories.
While control organoids had both rod and cone PRs, NRL null organoids had only cone PRs, with S-cones as the dominant subtype. 5144 control and 8173 NRL null PRs were subsetted from the rest of the retinal cell types in order to reconstruct the PR developmental trajectory specifically. The combined PR trajectory showed various possible cell fates, including two branches that contain control rods and S-opsin expressing NRL null PRs. Compared to the PRs along the cone developmental branch, these NRL null PRs have significantly lower expression of various cone-specific transducin and phosphodiesterase genes (p<10-8).
Consistent with the function of NRL in directing PRs towards a rod fate, retinal organoids lacking NRL developed cone-dominant PR populations. A subset of these cones are bioinformatically distinguishable due to their aberrant expression of transducin and phosphodiesterase genes, causing them to cluster with rod photoreceptors. Further analysis of these PR populations will better define the regulatory networks involved in PR differentiation and cell fate specification.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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