Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line

Abstract

Objectives: Cone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter.

Methods: We generated OPN1LW-EGFP/NRL^-/- mice by crossing NRL^-/- and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL^-/- mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL^-/- mice were transplanted subretinally into immunodeficient Rd1 mice.

Results: OPN1LW-EGFP/NRL^-/- retinas were enriched with OPN1LW-EGFP⁺ and S-opsin⁺ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL^-/- compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL^-/- were similar as the NRL^-/- with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL^-/- was thinner than NRL^-/- controls. The OPN1LW-EGFP/NRL^-/- ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL^-/- controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL^-/- mice showed greater S-opsin ⁺ cone survival than suspension grafts from the same strain.

Conclusions: OPN1LW-EGFP/NRL^-/- retinae were enriched with S-opsin⁺ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin⁺ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Keywords: Age-related macular degeneration; Cone photoreceptor; Cone transplantation; Degenerative retinal diseases; Optic coherence tomography; Stem cell therapy.

Fig. 1.. Characterization of cone photoreceptors in OPN1LW-EGFP/NRL^−/− mice by SWF imaging and immunohistochemistry (IHC).

(A) SWF imaging showed the density and topography of OPN1LW-EGFP⁺ photoreceptors in the fundus of OPN1LW-EGFP/NRL^−/− mice and control mice (OPN1LW-EGFP, NRL^−/−, C57BL/6J mice). (B) Retinal flat mount of OPN1LW-EGFP/NRL^−/− mice (P36) and age-matched control mice were stained with L/M-opsin (L/M-cone) and S-opsin (S-cone). Anti-GFP-FITC was used to highlight OPN1LW-EGFP⁺ cells. Representative images showed the morphology and distribution of L/M- and S-cone subpopulations in both dorsal- and ventral- retina. (C) Quantification of OPN1LW-EGFP, L/M-opsin, and S-opsin density in retinal flat-mounts of OPN1LW-EGFP/NRL^−/− and control mice. The heatmap shows the density topography of cone-specific markers (OPN1LW-EGFP, L/M-opsin, S-opsin) in the strains studied. (D) Colocalization analysis using IHC staining of GFP-FITC and S-opsin. (D1) retinal sections from OPN1LW-EGFP/NRL^−/− mice (P7, P10, P15, and P35) showed the co-localization of S-opsin and OPN1LW-EGFP expression (magnified in the upper left corner of each panel). Co-localization frequency of S-opsin and OPN1LW-EGFP was quantified by % of ROI colocalized. Fractions of OPN1LW-EGFP and S-opsin signal in colocalized compartments were analyzed via MCC (MCC_G and MCC_R, respectively); (D2) Schematic presenting the imaging regions of interest (ROIs) on retinal flat mounts from OPN1LW-EGFP/NRL^−/− and OPN1LW-EGFP mice (P35); (D3) Representative 3D-images showed the colocalization of OPN1LW-EGFP and S-opsin on retina flat mounts (magnified on the right for each panel); (D4) Spots colocalization counting of % cones that co-expressed OPN1LW-EGFP and S-opsin (EGFP⁺S⁺), or purely expressed OPN1LW-EGFP (EGFP⁺S⁻) or S-opsin (EGFP⁻S⁺). Abbreviations: SWF: short-wavelength fluorescence; ROI: region of interest; MCC: Mander’s colocalization coefficient.

Fig. 2.. Rosette development and retinal thinning in OPN1LW-EGFP/NRL^−/− mice.

(A) MR imaging and SD-OCT scanning of OPN1LW-EGFP/NRL^−/−, OPN1LW-EGFP, NRL^−/−, and C57BL/6J (wild-type) mice. White arrows: representative rosettes. (B) Retina sections stained with S-opsin showed rosette development in OPN1LW-EGFP/NRL^−/− and NRL^−/− mice (from P7 to P35~P40). There were comparable numbers of mature rosettes in the OPN1LW-EGFP/NRL^−/− and NRL^−/− mice during development, except for at P15 where the number of rosettes in OPN1LW-EGFP/NRL^−/− were higher (*p = 0.015). Data were collected from both the dorsal and the ventral retina. White arrows: representative nascent rosettes; Yellow arrows: representative mature rosettes. (C) Whole retina thickness was computed on SD-OCT B-scans across the different retinal regions (0.5 μm, 427.2 μm, and 854.4 μm from the optic nerve). The thickness of photoreceptor inner and outer segments (IS/OS), the outer retina (ONL/OLM), and the inner retina (OPL, INL, IPL, RGC) were quantified at a distance of 427.2 μm in the dorsal retina. * p < 0.0001. Abbreviations: MR: multicolor reflectance; IR: infrared; SD-OCT: spectral domain optical coherence tomography; INL: inner nuclear layer; ONL: outer nuclear layer.

Fig. 3.. Cellular composition of rosettes in OPN1LW-EGFP/NRL^−/− mice.

(A) IHC images of P35 OPN1LW-EGFP/NRL^−/− retinal sections stained with retinal specific markers, including S-opsin, L/M-opsin, Rhodopsin, GFAP, and PKC-α, with age-matched NRL^−/− retina as control. (B) Three-dimensional reconstitution of S-opsin⁺ and L/M-opsin⁺ rosette on retina flat mount of OPN1LW-EGFP/NRL^−/− mice (P35). (C) Quantification of rosettes that were positive for each marker in OPN1LW-EGFP/NRL^−/− and NRL^−/− mice. * p < 0.0001. NS: not significant. (D) Transmission electron microscopy of a representative rosette in OPN1LW-EGFP/NRL^−/− mice (P35). The magnified image is on the right side. Abbreviations: RGC: retinal ganglion cell; INL: inner nuclear layer; ONL: outer nuclear layer; OLM: outer limiting membrane; IS: inner segment; CC: connecting cilium; OS: outer segment.

Fig. 4.. Retinal function tested by full-field electroretinography (ERG).

(A) Representative ERG responses of OPN1LW-EGFP/NRL^−/− mice and control mice (OPN1LW-EGFP, NRL^−/−, C57BL/6J mice). Scotopic-, photopic-, 465 nm-, and 525 nm-stimuli ERG were performed to evaluate the function of rods, cones, S-cones, and M-cones, respectively. All waveforms start at 0 millisecond (ms). Stimulus intensity was 0.6 log cd.s/m² in each case. (B) Comparison of the full-field ERG response amplitudes between OPN1LW-EGFP/NRL^−/− and control mice over an intensity response range. Statistical analysis: Scotopic a-wave: * p = 0.016 (OPN1LW-EGFP/NRL^−/− vs C57BL/6J), ** p = 0.021(OPN1LW-EGFP/NRL^−/− vs OPN1LW-EGFP), *** p < 0.0001. Scotopic b-wave: * p < 0.0001. Photopic b-wave: * p = 0.028, ** p = 0.008, *** p < 0.0001. 465 nm stimuli b-wave: * p = 0.002, ** p < 0.0001. 525 nm stimuli b-wave: * p = 0.035, ** p = 0.0001, *** p < 0.0001. Comparison of 465 nm and 525 nm b-wave amplitude in OPN1LW-EGFP/NRL^−/−mice: * p = 0.0006, ** p < 0.0001.

Fig. 5.. Evaluation of grafted OPN1LW-EGFP/NRL^−/− retinal suspensions and sheet grafts in Rd1/NS recipients.

(A) Multimodal cSLO images from representative Rd1/NS recipients and PBS mock transplanted Rd1/NS control mice. SWF and SD-OCT imaging were used to track the grafted retinal sheet and suspension from OPN1LW-EGFP/NRL^−/− mice at 1 month and 3 months post-transplantation. Between white arrows: recipient retina; Between yellow arrows: retina grafts. (B) The mean change ratios of graft area and intensity from 1 month to 3 months were compared between sheet and suspension grafts on SWF imaging. The mean thickness change ratio of sheet and suspension grafts over three months was quantified on SD-OCT images. For sheet versus suspension grafts: Area change ratio: 1.49 ± 1.12 vs. 0.25 ± 0.48, n =12, p =0.002; Intensity change ratio: 0.76 ± 0.53 vs. 0.21 ± 0.32, n =12, p = 0.006; Thickness change ratio: 0.92 ± 0.12 vs. 0.42 ± 0.30, n =18, p <0.0001. (C) IHC staining of OPN1LW-EGFP and cone-specific markers (L/M-opsin, S-opsin) was performed on sheet and suspension grafts (C1–C2, C4–C5)), and PBS-transplanted controls (C6), at 3 months post-transplantation and P6 OPN1LW-EGFP/NRL^−/− donor retina controls (C3). Asterisk: neurite of a grafted cone photoreceptor. Scale bar = 20 μm. (D) Quantification of grafted cone subpopulations expressing OPN1LW-EGFP, L/M-opsin, and S-opsin in retinal sheet graft, suspension graft, and the P6 OPN1LW-EGFP/NRL^−/− donor retina sections. Abbreviations: SWF: short-wavelength fluorescence; MR: multicolor reflectance; IR: infrared; SD-OCT: spectral domain optical coherence tomography; RGC: retinal ganglion cell; INL: inner nuclear layer.