September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Gene-edited human induced pluripotent stem cell derived 3D retinas for the systematic optimization of retinal cup formation by small molecule screening.
Author Affiliations & Notes
  • Karl J Wahlin
    Ophthalmology, University of California, San Diego, LaJolla, California, United States
  • Srinivas Rao Sripathi
    Ophthalmology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Kiara Eldred
    Biology, Johns Hopkins University, Baltimore, Maryland, United States
  • Jacob Heng
    Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Robert Johnston
    Biology, Johns Hopkins University, Baltimore, Maryland, United States
  • Cynthia Berlinicke
    Ophthalmology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Donald J Zack
    Ophthalmology, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Karl Wahlin, None; Srinivas Sripathi, None; Kiara Eldred, None; Jacob Heng, None; Robert Johnston, None; Cynthia Berlinicke, None; Donald Zack, None
  • Footnotes
    Support  NEI Grant EY024648
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Karl J Wahlin, Srinivas Rao Sripathi, Kiara Eldred, Jacob Heng, Robert Johnston, Cynthia Berlinicke, Donald J Zack; Gene-edited human induced pluripotent stem cell derived 3D retinas for the systematic optimization of retinal cup formation by small molecule screening.. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Retinal degenerative diseases, such retinitis pigmentosa (RP), cause photoreceptor (PR) cell death and blindness. There are no cures and significant gaps exist in our understanding of how PR loss occurs. To address this we have developed genetically modified human induced pluripotent stem cell (hiPSC) retinal cell-reporters to investigate pathways that could lead to more efficient generation of human 3D retinas in vitro.

Methods : This work bridges several overlapping technologies; (1) hiPSCs to generate 3D retinas, (2) gene-editing using CRISPR technology to generate retinal reporter hiPSCs and (3) a small molecule chemical screen to generate more efficient retinas. We adapted the forced aggregate approach pioneered by Nakano et al (2010) for embryoid body formation and could generate optic vesicles after 10 days invitro. To generate retinal reporters, hiPSCs were transfected with guide RNAs, a Cas9 nuclease, and a P2A-GFP (FxP) targeting cassettes specific for the early retina gene SIX6. In addition to manipulating the cellular microenvironment, small molecules from the Stem Select Library were administered at multiple doses and 3D optic vesicles and assayed for reporter expression using an automated microscope 96-well based imaging system.

Results : Using the CRISPR gene-editing approach we successfully modified several retinal genes thereby generating reporters for the early etinal marker SIX6 among others. Expression of GFP was directly linked to modifications to the microenvironment and well as Shh signaling. Analysis of small molecule treated SIX6 reporter cells, which recapitulate early retinal expression of SIX6, have uncovered several players that appear to partcipate in early human retinal cup formation in vitro. The 3D retinas derived from this method bore complex laminar morphologies and rod and cone photoreceptor outer segment-like structures.

Conclusions : A main goal of our work was to identify novel mechanisms for retinal development that could increase the efficiency and pace of PR generation and provide insight into human ocular development. These reporter cell lines thus offer a potentially robust platform for the study of photoreceptor commitment and maturation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

SIX6-GFP expressing 3D embryoid bodies generated using an optimized retinal induction protocol.

SIX6-GFP expressing 3D embryoid bodies generated using an optimized retinal induction protocol.

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