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Adam Miltner, Stephen Kwong, Simranjeet Cheema, Yesica Mercado-Ayon, Rose Dixon, Anna La Torre; Comparing mouse retinal organoid differentiation to mouse retina development. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3326.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell derived Retinal Organoids (ROs) are a widely used tool for studying complex developmental and disease mechanisms. While we know that pluripotent cells can self-organize into a laminated retinal tissue, it is not clear how faithfully ROs replicate characteristics of the normal retina. Here, we focused on stem cell-derived retinal ganglion cells (SC-derived RGCs) and how they compared to endogenous RGCs in the mouse retina. Specifically, we studied whether the expression of three miRNAs—let-7, miR-9, and miR-125b (late progenitor or LP-miRNAs) act to temporally limit the production of RGCs, as they do in retinal development. We also compared physiology and gene expression profiles between endogenous RGCs and SC-derived RGCs to gain a better understanding of similarities and differences between RGCs in a retina vs in an organoid.
Induced Pluripotent Stem Cells (iPSCs) were made from two RGC reporter mice: Isl2-GFP and Brn3b-mCherry. These iPSCs were then differentiated into ROs. EdU lineage tracing was used to identify a potential competence window for RGC production in vitro. Quantitative PCR was used to measure LP-miRNA levels in ROs. A perforated patch technique was used to measure action potentials from RGCs, and a Calcium sensing dye used for Calcium imaging. RNA sequencing (RNAseq) was performed by the UC Davis sequencing core facility. In electrophysiology and RNAseq experiments, RGCs derived from mouse retinas were used as control samples.
Combining EdU lineage tracing with our RGC reporter lines, we found that RGC production in ROs was limited from differentiation day 8 to day 14. Additionally, we found that LP-miRNAs increased during the same differentiation time. In our efforts to further compare SC-derived RGCs to endogenous RGCs, we analyzed SC-derived RGC physiology and found that SC-derived RGCs were capable of firing action potentials. Finally, by RNAseq, we determined that SC-derived RGCs made from our RGC reporter lines expressed many of the same genes as their cognate RGCs produced in the mouse retina.
We conclude that ROs mimic the temporal restrictions on RGC production found in development, and uncovered evidence suggesting this temporal competence is miRNA-mediated. Additionally, we found that SC-derived RGCs expressed many of the same genes found in endogenous RGCs. This work highlights the usefulness of using ROs in the place of animal models.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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