June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
A novel retinal ganglion cell reporter stem cell line for timing neurogenesis in retinal organoids
Author Affiliations & Notes
  • Adam Miltner
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Stephen Kwong
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Yesica Mercado-Ayon
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Simranjeet K. Cheema
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Tom Glaser
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Anna La Torre
    Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Footnotes
    Commercial Relationships   Adam Miltner, None; Stephen Kwong, None; Yesica Mercado-Ayon, None; Simranjeet Cheema, None; Tom Glaser, None; Anna La Torre, None
  • Footnotes
    Support  NIH Grants R01EY026942 awarded to Anna La Torre and 4T32EY015387 awarded to Adam Miltner
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1677. doi:
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      Adam Miltner, Stephen Kwong, Yesica Mercado-Ayon, Simranjeet K. Cheema, Tom Glaser, Anna La Torre; A novel retinal ganglion cell reporter stem cell line for timing neurogenesis in retinal organoids. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1677.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Stem cell-derived retinal organoids are widely used to study complex developmental and disease mechanisms. Pluripotent cells can self-organize to form laminated retinal tissue in vitro, but it is unclear whether organoids follow the same histogenetic clock or molecular mechanisms as retinal progenitors in vivo. As a first step to improve organoid disease modeling and use in future therapy we charted the timing of retinal ganglion cell (RGC) neurogenesis in organoid cultures and tested the role of three late-progenitor micro RNAs (LP-miRNAs)—let-7, miR-9, and miR-125b—in retinal organoid differentiation in vitro.

Methods : We isolated induced Pluripotent Stem Cells (iPSCs) from Isl2-GFP BAC transgenic RGC reporter mouse embryonic fibroblasts by transduction with Nanog, Lin28, Sox2, and Oct4. To determine RGC birthdates, we added 5-ethynl-2’-deoxyuridine (EdU) to organoid cultures during four different two-day windows, and we measured LP-miRNA levels over time by quantitative PCR. To inhibit LP-miRNAs, we stably targeted Isl2-GFP iPSCs with an inducible poly-sponge vector via CRISPR/Cas9 recombination.

Results : iPSC-derived RGCs expressing Isl2-GFP were born from differentiation days 7 to 15 with peak birthdates at day 10. These EdU labeling results suggest that LP-miRNAs may act to limit RGC genesis over time, as they do in vivo during normal retinal development. To directly test how LP-miRNAs affect organoid RGC production, we isolated Isl2-GFP iPSC clones that express a polymerized miRNA sponge under the control of a Tet-On system. The multivalent sponge targets all three LP-miRNAs, is incorporated into the 3’ UTR of an RFP fluorescent reporter, and is strongly induced after doxycycline addition.

Conclusions : Retinal organoids possess great potential as therapeutic and disease model tools. The timing of RGC birthdates match in vitro and in vivo—and appear to be controlled by the same miRNA mechanisms, providing a basis to model disease and manipulate organoids to produce specific cell types or molecules.

This is a 2021 ARVO Annual Meeting abstract.

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