Generation of a Cone Photoreceptor-specific GNGT2 Reporter Line in Human Pluripotent Stem Cells

Abstract

Fluorescent reporter lines generated in human pluripotent stem cells are a highly useful tool to track, isolate and analyse cell types and lineages in live cultures. Here, we generate the first human cone photoreceptor reporter cell line by CRISPR/Cas9 genome editing of a human embryonic stem cell (hESC) line to tag both alleles of the Guanine nucleotide-binding protein subunit gamma-T2 (GNGT2) gene with a mCherry reporter cassette. Three-dimensional optic vesicle-like structures were produced to verify reporter fidelity and track cones throughout their development in culture. The GNGT2-T2A-mCherry hESC line faithfully and robustly labels GNGT2-expressing cones throughout the entirety of their differentiation in vitro, recapitulating normal fetal expression of this gene. Our observations indicate that human cones undergo significant migratory activity during the course of differentiation in vitro. Consistent with this, our analysis of human fetal retinae from different stages of development finds positional differences of the cone population depending on their state of maturation. This novel reporter line will provide a useful tool for investigating human cone development and disease.

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model

“…Human photoreceptor– and rod-specific embryonic stem cell/iPSC (ESC/iPSC) reporter lines have been previously generated ( 22 , 24 – 27 ), and a very recent study produced a cone-specific human ESC line ( 28 ), however, to our knowledge, no cone reporter hiPSC line has been reported. Based on immunohistochemical and transcriptional assessment, the mCar-GFP iPSC reporter line presented here appeared to robustly and specifically label human cone photoreceptors.…”

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids

“…Recently, two other cone reporter human iPSC lines have been described -one generated by piggyBac mediated insertion of GFP under the control of mouse cone-arrestin (mCar) promoter (Gasparini et al, 2022) and the other generated by inserting a T2A-mCherry cassette into the GNGT2 locus (Nazlamova et al, 2022).…”

“…Recently, two other cone reporter human iPSC lines have been described - one generated by piggyBac mediated insertion of GFP under the control of mouse cone-arrestin (mCar) promoter (Gasparini et al, 2022) and the other generated by inserting a T2A-mCherry cassette into the GNGT2 locus (Nazlamova et al, 2022). Cone-arrestin is first expressed at a later stage of cone maturation than GNAT2, limiting its ability to label immature cones (Hoshino et al, 2017; Welby et al, 2017) (Fig.…”

“…S1O) (Lu et al, 2020) (Shayler et al, in revision ). Nevertheless, GNGT2 protein is reported to be specifically expressed in cones of the developing and adult human retinae (Ong et al, 1997) as is mCherry protein in GNGT2-T2A-mCherry ROs (Nazlamova et al, 2022), suggesting that post-transcriptional mechanisms enforce cone-specific translation of GNGT2 and the linked mCherry in GNGT2-T2A-mCherry ROs, despite similar cone and rod GNGT2 RNA levels.…”

“…Photoreceptor reporter lines have been generated by introducing a CRX- GFP cassette (Kaewkhaw et al, 2015) or a mouse Crx-mCherry cassette (Gagliardi et al, 2018) into the AAVS1 locus. A rod reporter line was made by replacing the NRL coding sequence with EGFP (Phillips et al, 2018); cone reporter lines were produced by inserting a mouse cone-arrestin (mCar)-GFP cassette (Gasparini et al, 2022) or inserting T2A-mCherry at the C terminus of GNGT2 (Nazlamova et al, 2022); and a retinal ganglion cell (RGC) reporter line was produced by inserting a P2A-tdTomato-P2A-Thy1.2 cassette at the C terminus of BRN3B (Sluch et al, 2017). Additional lines reporting multiple cell types include a SIX6 -GFP / POU4F2 -tdTomato double reporter line that separately labels all retinal cells and RGCs and a VSX2 -Cerulean / BRN3b -EGFP / RCVRN -mCherry triple reporter line that differentially labels retinal progenitor cells, RGCs, and photoreceptors (Lam et al, 2020; Wahlin et al, 2021).…”