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Ziming Luo, Kun-Che Chang, Bogdan Tanasa, Jeffrey L Goldberg; Understanding RGC differentiation and development in retinal organoids by scRNA-seq. Invest. Ophthalmol. Vis. Sci. 2022;63(7):4501 – F0288.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) replacement therapy could provide an approach to vision restoration in glaucoma and other optic neuropathies. RGCs generated from retinal organoids are promising donor cells. The present research aims to understand how organoid RGCs (oRGCs) differ from human fetal RGCs.
We generated retinal organoids from Brn3b-tdTomato-H9 hESC line. Then, we utilized single cell-RNA sequencing (scRNA-seq) to delineate the oRGC differentiation and compare their transcriptomic profiles to human fetal RGCs. EdU incorporation assays were used to visualize the newly born oRGCs in retinal organoids of different developmental stages. All experiments were conducted at least three times independently. Data were analyzed by ANOVA and post-hoc t-test with Tukey correction, with a P-value of <0.05 considered statistically significant.
At early developmental stages, oRGCs had similar expression patterns to fetal RGCs in corresponding timepoints. By UMAP plotting and pseudotime analyses, we found oRGCs overlapped with fetal RGCs at earlier time points. However, with ongoing development, RGC numbers decreased rapidly in retinal organoids, and their transcriptomes diverged from fetal developmental patterns. Transcripts from neurofilament genes were relatively low and did not increase along developmental time in oRGCs, but were highly expressed in adult RGCs. Moreover, POU4F2 (Brn3b) expression in oRGCs was maintained at a high expression level for a longer time, while in fetal RGCs, Brn3b was significantly downregulated with maturation. Interestingly, RBPMS, an RGC-specific marker, was barely expressed in organoid RGCs. EdU incorporation assays revealed that oRGC generation slowed down around the 10th week of differentiation, coming nearly to a complete halt by the 15th week in retinal organoids.
In the present study, we describe the transcriptomic differences between oRGCs and fetal RGCs, providing substantial insight into towards stem cell-derived RGC development, towards enhancing our understanding of stem cell differentiation and in the future, stem cell-based therapeutic interventions towards restoring vision in human disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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