Fig 1: Thy1-GFP+ donor RGCs survive and are traceable in vivo post-transplantation(A) RGC cell replacement pipeline. Transplants were performed using Thy1+ donor cells isolated with magnetic microbeads for CD90.2, delivered at 10,000 cells/μL. ∗For RNA sequencing, RGCs were isolated using L1cam (CD171). (B) Characterization of Thy1-GFP+ donor cell population within organoids. 50% of Thy1-expressing cells also expressed the Thy1-GFP+ transgene; near 100% of Thy1-GFP+ cells expressed RBPMS, a pan-RGC marker. Hence, for transplant assessment we assume that only 50% of transplanted donor RGCs will be detectable by GFP expression and that GFP+ cells are all RGCs. Thy1-GFP− cells will not be traceable due to the allogeneic nature of the transplant. (C) In vivo fundus imaging of intravitreally delivered Thy1-GFP+ donor cells in anesthetized mice at 2 weeks post-transplantation. Thy1-GFP+ cells were found throughout the vitreous cavity and toward the back of the eye, ectopic to the retina. Intrinsic Thy1-GFP expression of donor RGCs was sufficient to allow for in vivo resolution of axons and smaller, neuronal processes. (D) Post-enucleation and fixation, Thy1-GFP+ donor RGCs are detectable within retinal flat mounts displaying diverse neuronal morphology as early as 2 weeks post-transplant.
Fig 2: Thy1-GFP iPSC-derived organoids contain molecularly diverse RGCs at day 21(A) Simplified hierarchy of known RGC-specific markers delineating mouse RGC subtype diversity according to previously published data. (B) Confocal imaging setup for whole retinal organoids. Three-week-old retinal organoids are suspended within concavity slides using PBS and sealed with a cover glass during confocal microscopy. Avoiding the use of solidifying mounting media enables the flexible rotation of the sample during imaging to enable efficient capture of Thy1-GFP+ areas. (C–F) While Thy1-GFP+ cells overlap with several pan-RGC markers, including RBPMS, Brn3a, Thy1, and Brn3c, as indicated by asterisks, Brn3a and RBPMS can be seen to also capture non-GFP-expressing cells, underlining the mosaic nature of the Thy1-GFP transgene. (G–K) Aside from several pan-RGC markers, a comprehensive set of RGC subtype-specific markers was detected including: osteopontin, Tbr1, CART, melanopsin, and FoxP1, pointing to the presence of several RGC subgroups including J-RGCs, alpha/intrinsically photosensitive RGCs (ipRGCs), and direction-selective ganglion cells (DSGCs). Asterisks highlight some of the double-positive cells.
Fig 3: RGC numbers are not altered in either WT or P2X7-KO retinae following acute IOP elevation. RGCs were immunohistochemically labelled with RBPMS and images of representative staining of the central (A) and peripheral (B) eccentricities are shown. RGC counting showed no difference in RGC number between central (C) and peripheral (D) eccentricities. Numbers in histogram indicate number of animals. Scale bar 20 µm.
Fig 4: Reliable detection of residual visual acuity in mice with severe optic nerve damage. (A) Schematic illustration of optic nerve crush (ONC). The right optic nerve was pinched with tweezers behind the globe. (B) Histological sections with Hematoxylin-Eosin staining of the optic nerve, untouched and after ONC (right and left, respectively). Note that ONC destroyed the tissue and led to the infiltration of inflammatory cells. Scale bar: 50 μm. (C) Histological sections of the retinal ganglion cell (RGC) layer in an ONC eye. Rbpms-positive RGCs (green) were fewer 7 days after ONC. Scale bar: 20 μm. (D) OKR-measured visual acuity in the ONC model. Optomotry could not detect residual visual acuity after ONC (ONC + (R)). Note that measured visual acuity after ONC is comparable to the background noise level of the system (Neg. Cont.; P = 0.883. N = 10 for untouched mice (untouched (R)), N = 10 for ONC mice (ONC – (L) and ONC + (R)), and N = 5 for Neg. Cont. *P < 0.05, ***P < 0.001. (E) Representative pVEP traces recorded from the hemisphere contralateral to the ONC eye. pVEP from an untouched mouse and a blinded control (both eyes were shielded) are also shown. Note that the pVEP waveforms in the ONC mouse were very different from the untouched mouse. (F) Summary of pVEP amplitudes measured in ONC mice, shown as a function of the spatial frequency of the pattern stimulus. N = 9 for ONC mice, N = 7 for untouched mice, and N = 4 for mice with both eyes shielded. Noise level = 4.67 μV (N = 4). (G) Comparison of visual acuity in ONC and untouched eyes, as evaluated with linear regression of pVEP amplitudes. Note that visual acuity in the ONC eyes is lower than the untouched eyes. ***P < 0.01., N = 9 for ONC mice, N = 7 for untouched mice. Data represent mean ± standard error of the mean. Neg. Cont.: negative control. (H) Comparison of visual acuity in ONC and untouched eyes, as evaluated with threshold determination of pVEP responses. ***P < 0.001. ANOVA followed by Tukey-Kramer’s multiple comparison tests was applied for comparisons. Data represent mean ± standard error of the mean.
Fig 5: The effect of COF delivered in vitro and in vivo. (A): The viabilities of N2A and BV2 cells, co-cultured with COF (0–1.34 μg/mL) for 6 and 24 h, were measured by CCK-8 assay kit (n = 6). (B): Quantitative PCR of IL-1β, TNF-α and iNOS in BV2 cells, pretreated with normal saline or COF (0.67 μg/mL), following stimulated by LPS (1 μg/mL) and IFN-γ (100 ng/mL) for 18 h (n = 3). (C): Western blotting and statistical graph of BCL2/BAX of retinas after intravitreal injection normal saline and COF. (D): Representative immunohistochemical images and statistical graphs of the number of RBPMS-positive RGCs in retinas three and six weeks after single intravitreal injection COF. (n = 4). Scale bar = 50 um. * indicates p < 0.05, ** indicates p < 0.01. (E): Representative immunohistochemical images of anti-GFAP and anti-Iba1 in retinas one to six weeks after single intravitreal injection COF. (n = 4).
Supplier Page from Abcam for Anti-RBPMS antibody