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Melissa Kaye Jones, Anna R Ogata, Fei Su, Bolin Chen, Justin Seid, Karl Wahlin; Transcriptomic analysis of retinal organoids from human pluripotent stem cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):574. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The use of pluripotent stem cells (PSCs) to study human eye development and disease has been facilitated by new gene editing technologies that enable tracking of cells during development and the course of degeneration. The differentiation of human PSCs into three-dimensional organoids using reporter cells allows for the study of human retinogenesis in real time. Although the molecular events underlying this process is not yet complete, cell fate specification and maturation follows a sequential time course with some known markers for retinal progenitor cells. Markers for retinal progenitors, including Six6 and Vsx2, that appear at different stages of development could be used to further elucidate the onset of retinal induction and cell fate commitment. Transcriptomic analysis of key developmental time-points could also aid in the identification of additional biomarkers.
The CRISPR-Cas9 system was used to create a Six6-GFP and Vsx2-Tomato dual reporter. hPSCs were transfected and genotyped for the Six6-GFP and Vsx2-Tomato constructs. hPSCs were differentiated into three-dimensional organoids, and selected for Six6-GFP and Vsx2-Tomato positivity. Organoids were collected at different time-points during development, and transcriptomic studies were performed.
Organoids expressed GFP+ and Tomato+ signals indicating expression of Six6 and/or Vsx2 during development. Transcriptomic analysis identified gene expression changes that occur during the transition of an early Six6-GFP+ eye field progenitor to a Vsx2-Tomato+ retinal progenitor. Bioinformatic analysis identified signaling pathways that may play a role in retinal differentiation.
A dual reporter for Six6 and Vsx2 expression in three-dimensional retinal organoids was developed. Key gene expression changes occur with retinal induction and maturation. In addition, signaling pathways that play roles in retinal differentiation were identified. Further analyses of these mechanisms will lead to better understanding of gene regulation during retinal development.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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