Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types
2019
Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retina progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist. Here, we describe a CRISPR/Cas9 gene editing strategy to generate a triple transgenic reporter hiPSC line (PGP1) that utilizes the endogenous VSX2, BRN3b, and RCVRN promoters to specifically express fluorescent proteins (Cerulean in NRPs, eGFP in RGCs and mCherry in PRs) without disrupting the function of the endogenous alleles. Retinal organoid formation from the PGP1 line demonstrated the ability of the edited cells to undergo normal retina development while exhibiting appropriate fluorescent protein expression consistent with the onset of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types. The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.
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