Abstract
Purpose :
Photoreceptor precursor cell transplantation is an attractive strategy to regenerate photoreceptors following retinal degeneration. However, current approaches are limited by poor donor cell integration and functional recovery. We hypothesize that the cellular heterogeneity of transplanted cells may impair their integration potential, and performed unbiased transcriptomic analysis of the developing mouse retina to identify an optimal photoreceptor precursor cell population for subretinal transplantation.
Methods :
Single-cell RNA-sequencing was performed throughout a time course of normal murine retinogenesis to reveal subpopulations of Crx+ photoreceptor precursor cells. Surface markers were identified for each subpopulation and validated by RNA fluorescent in situ hybridization (FISH), immunofluorescence staining and qPCR. Fluorescently labeled donor cells were purified from postnatal day 3-4 Tg(CAG-eGFP); RosanT-nG transgenic mice via papain dissociation and fluorescence activated cell sorting (FACS). 300,000 donor cells were injected subretinally into the Lebercilin (Lca5)-deficient mouse model of retinal degeneration. Cellular location and morphology was assessed two-months post-transplantation by in vivo imaging, histology, and immunofluorescence staining.
Results :
Transcriptomic analysis of the neonate murine retina revealed three distinct subpopulations of Crx+ cells consistent with early (Dll1+), intermediate (Neurod4+), and late (Prom1+) photoreceptor precursor cells. These populations have distinct transcriptional and spatial expression patterns in the early postnatal mouse retina. Two months post-transplantation in the Lca5-/- mouse, fluorescently labeled Prom1+ cells survive, are detectable in the subretinal space, and generate robust projections compared to unsorted cells.
Conclusions :
There is transcriptional heterogeneity of developing photoreceptor cells in the neonatal mouse retina. Prom1+ cells produce robust cellular projections following subretinal injection in the Lca5-/- mouse, suggesting that transplantation of an optimal developmental stage of photoreceptor precursor cells may improve regenerative strategies. Further studies are needed to evaluate the potential of these populations to differentiate into mature functional photoreceptors.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.