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.