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Joe Phillips, Jee Min, Sara Howden, Peng Jiang, Li-Fang Chu, Patrick Barney, Anna Petelinsek, Elizabeth E Capowski, Abbey Cash, David M Gamm; Characterization, single cell RNAseq, and transplantation analysis of neuroretinal cells derived from a human ESC-CRX reporter line. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3594.
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© ARVO (1962-2015); The Authors (2016-present)
We created a human embryonic stem cell (hESC)-derived CRX reporter line to interrogate photoreceptor differentiation in vitro and to identify transplanted photoreceptor precursors in vivo.
Gene targeting via homologous recombination was used to create hESC CRX:tdTomato lines, whereupon optic vesicle-like (OV)-like structures were generated using our established protocols. Individual hESC-OVs were imaged over time to track photoreceptor differentiation, followed by confocal microscopy on OV sections or high content analysis (Operetta) on dissociated, plated cells. For single cell RNAseq analysis, single cells were captured using the Fluidigm C1 system and sequenced (Illumina HiSeq 2500). Principal component, hierarchical clustering, and known marker gene expression analysis were performed to identify cell subpopulations. For transplantation analysis, dissociated hESC-OVs were injected subretinally into immune compromised (RNU) rats with normal or degenerative retinas (RNU-S334ter) and analyzed at 2 weeks post-transplant.
CRX expression was first detected at day 28; by day 60, >97% of OVs had CRX+ photoreceptors. ICC analysis showed extensive colocalization of tdTomato expression with CRX antibody and other photoreceptor markers. With time, photoreceptor precursors gave rise to both rods and cones as revealed by ICC. Single cell RNAseq analysis confirmed the retinal nature of cells within the OV, along with expression of genes from all major retinal cell classes. Evidence for cellular heterogeneity within each class was also seen, likely reflecting differences in maturation state. Lastly, transplanted neuroretinal cells derived from the reporter line, including tdTomato+ photoreceptor precursors, survived for at least 2 weeks in the rat subretinal space.
CRX expression in nearly every OV (and its absence in non-OVs) confirmed the robustness of our culture system, and single cell RNAseq analysis readily demonstrated the presence of multiple retinal cell subtypes within the differentiating cultures. These analyses will further guide studies aimed at transplanting hESC- and hiPSC-derived photoreceptors at optimal differentiation stage(s) to promote integration.
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