iPSC-Derived Retina Transplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice

Abstract

Recent success in functional recovery by photoreceptor precursor transplantation in dysfunctional retina has led to an increased interest in using embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal progenitors to treat retinal degeneration. However, cell-based therapies for end-stage degenerative retinas that have lost the outer nuclear layer (ONL) are still a big challenge. In the present study, by transplanting mouse iPSC-derived retinal tissue (miPSC retina) in the end-stage retinal-degeneration model (rd1), we visualized the direct contact between host bipolar cell terminals and the presynaptic terminal of graft photoreceptors by gene labeling, showed light-responsive behaviors in transplanted rd1 mice, and recorded responses from the host retina with transplants by ex vivo micro-electroretinography and ganglion cell recordings using a multiple-electrode array system. Our data provides a proof of concept for transplanting ESC/iPSC retinas to restore vision in end-stage retinal degeneration.

Keywords: iPSC; multiple electrode array; photoreceptor transplantation; retinal degeneration; retinal regeneration; shuttle-avoidance test.

Figure 1

Maturation of Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato Graft Retina (A and A′) Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato lines formed rhodopsin-positive ONL structures with outer-structure-like segments (asterisk) at DD35 after subretinal transplantation in rd1 mice, with (A) or without (A′) DAPI nuclear staining (transplanted at DD13). (B–B‴) tdTomato colocalized with anti-CtBP2 immunolabeling at the synaptic terminal of graft photoreceptors (DD35) (B). Merged images with and without DAPI (B, B′) of CtBP2 immunostaining (B″) and CtBP2-tdTomato visualization (B‴) with L7-GFP and Nrl-GFP. (C) CtBP2-positive synaptic terminals in the host retina that were negative for CtBP2-tdTomato (arrows). (D–D‴) tdTomato-positive graft synaptic terminals in the host retina at and around the tips of GFP-positive host bipolar dendrites (arrows); (D′) shows a side view of the sectional plane at the vertical dotted line in (D); (D″) shows a bottom view of the plane at the horizontal dotted line in (D); (D‴) shows CtBP2-tdTomato within the host retina was stained with the anti-CtBP2 antibody. Scale bars, 20 μm (A, C) and 10 μm (B, D).

Figure 2

Synaptic Integration of Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato Graft Retina into L7-GFP Host Mice (A) GFP-positive bipolar cells extend their dendrites through graft INL to reach CtBP2-tdTomato on graft ONL (DD78). (B) CtBP2-positive presynaptic terminals contact the tips of rod bipolar dendrite terminals in L7-GFP wild-type retina. Front view image of the section between the yellow dashed lines in side view, and side view image of the section between the yellow dashed lines in front view are presented. (C–C‴) 3D observation of contact between GFP-positive host bipolar cells and CtBP2-tdTomato in the graft ONL (DD35) with (C′) and without (C) DAPI nuclear stainings, with front (C″) and side slice views (C‴). (C″) is the image of the section between the yellow dashed lines in (C‴), and (C‴) is the image of the section between the two vertical lines in (C″). (D) CACNA1s localizes at dendritic tips of PKCα-positive dendrite terminals in a wild-type retina. (E and E′) CtBP2-tdTomato in the graft ONL (DD78) are coupled with CACNA1s (arrows) at the tips of L7-GFP-positive host bipolar cells as shown in the side and bottom sliced views. Scale bars, 20 μm (A, C), 10 μm (B), and 5 μm (D, E).

Figure 3

Shuttle-Avoidance Behavioral Tests in rd1 Mice with Retinal Transplants (A) The shuttle box has two compartments, a light in each compartment, and a beeping device in the middle. (B) An electric shock is delivered after 5 s of continuous light with or without beeping. (C) The experimental protocol for shuttle-avoidance testing (see Experimental Procedures). (D) Representative results for shuttle-avoidance tests using only light signaling for a wild-type (B6) mouse (left) and an rd1 (rd1-2J) mouse (right). The behavior of the wild-type mouse deviated significantly from that of an untreated control rd1 mouse, while that of the rd1 (rd1-2J) mouse did not. Dots denote the observed SAS test results (see also H) and the lines are estimated relationships of ITI count and SAS success count (30 trials in total) simulated from randomly selected posterior samples of the model; black and red indicate the control and the subject, respectively. (E) Posterior distributions of the estimated effect of strain difference or 9-cis retinol acetate administration to shock avoidance for rd1-2J and wild-type mice. White circles denote the median; bars denote the 95% confidence interval (CI) of the posterior distribution of β₃ for each animal (see also G). The 95% CI of all wild-type mice was above zero. (F) The number of mice that behaved differently from control rd1 mice in three groups: those with unsuccessful transplantation (N), a retinal transplant in one eye (M), and transplants in both eyes (B). (G) Posterior distributions of the estimated effect of transplantation to shock avoidance (β₃) for all mice that underwent SAS tests. (H) Representative SAS test results for individual mice (M8 and M10) with retinal transplants, with positive and negative results.

Figure 4

Ex Vivo MEA mERG and RGC Recordings from Transplanted Retinas (A) mERGs overlaid on a photograph of the isolated retina (TP-5 in Table 1) on the MEA microelectrodes. (B) RGC responses on each electrode in histograms after spike sorting. (C and D) Representative mERG (C) and RGC responses (D) in channels 15, 16 (orange box in A where the graft is thick), 25, and 26 (yellow box in A at graft margin) with or without the mGluR6 blocker. Black arrows indicate b-waves and red lines indicate timing of the signal flash (C). Blue arrows indicate transient ON responses and yellow bands indicate the timing and the duration of light stimuli (D). (E) A typical mERG wave pattern and RGC responses of a wild-type retina before and after L-AP4 treatment. After treatment of L-AP4, the b-wave in mERG and ON response (blue arrow) in RGC recording disappears.

Figure 5

Quantitative Analysis on RGC Spike Sources after Transplantation (A) RGC spike patterns among all of the detected spike sources in wild-type retinas (n = 2 retinas) with or without mGluR6 blocker, and in rd1 retinas with (n = 7) or without (n = 5) iPSC-retina transplants. (B) Details of RGC spike patterns in each retina sample after transplantation, clustered into each pattern shown in Figure S5A. Sample TP-7 is not shown because no spike source was clustered into any specific group. The number of detected spike sources is shown below each retina. (C) Number of RGC sources with typical light-responsive spikes in wild-type retina (transient ON/OFF and ON-OFF in Figure S5A) are plotted against the graft area indicated as the number of electrodes on x axis.

Figure 6

3D Histological Analysis of Retinal Transplants after MEA Recordings Tiled images of the retina after MEA (sample 5 in Table 1). The area on electrode channels 16 (A) and 25 (B) (orange box on MEA photograph) are shown with vertical section views. Green dotted lines indicate graft margin on the electrodes. Orange ovals indicate optic discs. Green arrows indicate L7-GFP-positive host bipolar cells in host INL (white arrows). CtBP2-tdTomato-positive graft synaptic terminals are present on the graft ONL margin (red arrows). (C) Magnified view of the section close to (A). Scale bars, 50 μm (A, B) and 20 μm (C).