Highlights
iPSC retina reconstructs outer nuclear layer in the end-stage retina
Contacts between the host bipolar cells and graft photoreceptors were visualized
rd1 mice became responsive to light after iPSC-retina transplantation
RGC responses to light were recorded from host rd1 retina after transplantation
Summary
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.
Introduction
Neonatal and fetal retina sheet transplants are reported to restore activity in host retinal ganglion cells or superior colliculus, a midbrain visual center for motor commands, in some mice and rats with retinal degeneration (Woch et al., 2001, Radner et al., 2001, Sagdullaev et al., 2003), and fetal retinas have been transplanted together with retinal pigment epithelium into patients with retinal degeneration and improved vision in some patients (Radtke et al., 2008). Mechanisms have been suggested as neurotrophic effect (Radner et al., 2001) or synaptic connections between unspecified inner retinal cells of host and graft (Seiler et al., 2008, Seiler et al., 2010), but there is no conclusive evidence that photoreceptors in these retina transplants can form functional synapses with host bipolar cells. Recently, Pearson et al. (2012) demonstrated that postnatal photoreceptor precursor cells can functionally integrate into the outer nuclear layer (ONL) of Gnat−/− mice and restore visual function; in these mice, rod photoreceptors are not functional but transplanted rod cells can restore scotopic visual function in a dose-dependent manner. Gonzalez-Cordero et al. (2013) then reported that mouse embryonic stem cell (mESC)-derived photoreceptor precursors can integrate into the ONL of mice retina. These reports, together with a number of reports describing protocols to differentiate retinal tissue from human ESCs or induced pluripotent stem cells (iPSCs) (Kuwahara et al., 2015, Nakano et al., 2012, Zhong et al., 2014), provided a basis for developing cell-based therapies for retinal degenerative diseases. More recently, however, Pearson’s and another group have reinterpreted their work, as new evidence has emerged that the functional restoration after photoreceptor transplantation had been more likely the result of biomaterial transfer from the transplanted cells to the local photoreceptor cells in the host ONL, rather than direct integration of the graft cells (Pearson et al., 2016, Santos-Ferreira et al., 2016). These reports brought us back to the initial question of whether the transplanted photoreceptors could make synapses with adult host retinal cells.
In clinical practice, cell-based therapies would primarily target end-stage retinas that have lost the ONL, leaving the secondary retinal neurons missing their partners for signal input. Therefore, the end-stage retinas can be considered to be in a different environment for graft cells from those of disease models retaining ONL that were used in the previous studies. Recent studies using cell suspensions of postnatal mouse photoreceptor precursors or human ES/iPS derived photoreceptor precursors in end-stage retinas, which have lost the ONL, indicated possible light response by pupillary reflex and behavior tests, although direct evidence of light response from the graft cells or synaptic function is still lacking. These graft photoreceptors did not develop mature morphology with outer segments or organized ONL structure that is important for photoreceptors to efficiently respond to light (Barnea-Cramer et al., 2016, Singh et al., 2013). In addition, rat fetal retina sheet grafts (Sagdullaev et al., 2003) apparently survive longer than mouse cell suspensions (Mandai et al., 2012, West et al., 2010), and a fetal retinal graft sheet in a clinical trial was observed to survive 3 years after the transplantation, while transplants in the form of microaggregates were no longer detected (del Cerro et al., 2000). Reconstruction of a structured ONL would definitely be ideal in these cases, but it has not been clearly demonstrated that an ES/iPS-derived structured, retina-like sheet can restore visual function. The difficulty of proving that visual function is present in mice and rats adds to the challenge of developing effective therapies for retinal degeneration.
We previously reported that 3D retinal tissue differentiated from mESC or mouse iPSC (miPSC) retina can develop a structured ONL of fully mature photoreceptors with outer-segment structures when transplanted into rd1 end-stage retinal-degeneration model mice, possibly by forming synapses with host bipolar cells (Assawachananont et al., 2014). These grafts can integrate with or without the presence of graft inner cells between the host inner nuclear layer (INL) and the graft ONL; the latter most closely resembles the natural retina and appears most promising for restoring visual function, although this has not been demonstrated. More recently, we also showed that hESC retina can similarly develop and mature after transplantation in the ONL-lost retinal-degeneration models of rats and monkeys in a similar manner (Shirai et al., 2015). Thus, the current question is whether the integrated ONL responds to light and transmits signals to host upper neurons in the end-stage host retina, especially to the retinal ganglion cells (RGCs) that subsequently transmit the neural signals to the brain.
In the present study, we assessed the visual function in rd1 mice with miPSC-retina transplants using a refined protocol on shuttle-avoidance tests, analyzed the light-responsive function in retinal transplants using ex vivo micro-electroretinogram (mERG) and RGC recordings, and examined the host-graft contact directly by genetically labeling the graft photoreceptor terminals. Our data provide a proof of concept for transplanting ESC/iPSC retinas to treat end-stage retinal degeneration. Here we also demonstrate practical, reliable methods for qualitatively and quantitatively assessing visual function in regenerative studies in mice.
Results
Visualization of Direct Contact between Host Bipolar Cells and Synaptic Terminals of Graft Rod Photoreceptors
To clearly identify direct host-graft integration, we generated iPSC lines that express CtBP2-tdTomato at photoreceptor synaptic terminals after differentiation (Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato) (Figures S1A–S1C). We also prepared an end-stage retinal-degeneration model mouse that expresses GFP in rod bipolar cells (L7-GFP/rd1) by crossing rd1-2J and L7-GFP mice. We found that three of the Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato lines differentiated to form optic vesicles similar to those we described previously (Figure S1D). A retinal sheet cut out from an optic vesicle on differentiation day 13 (DD13) was transplanted into each L7-GFP/rd1mouse; after transplantation, the grafts developed an Nrl-GFP-positive, rhodopsin-positive ONL with outer-segment-like structures on DD35, as we showed previously, although GFP positivity was weaker compared with later DDs (Figures 1A and 1A′ ) (Assawachananont et al., 2014), and L7-GFP-positive host bipolar terminals contacted CtBP2-tdTomato-positive graft regions. CtBP2-tdTomato, which was present along the margins of the Nrl-GFP-positive graft ONL, colocalized with anti-CtBP2 immunostaining (Figures 1B–1B‴). CtBP2-tdTomato also clustered at the tips of graft bipolar dendrites where they formed intra-graft synapses with Nrl-GFP-positive photoreceptors (Figure S2A). These findings indicate that tdTomato represents the CtBP2 expression at graft photoreceptor terminals. Some terminals in the host retina were negative for tdTomato (Figure 1C, arrows), suggesting that they were the remnants of host photoreceptors. We also found the outgrowth of tdTomato-positive graft CtBP2 into the host synaptic layer (Figures 1D–1D‴, arrows). We then more closely studied the contact between L7-GFP-positive host bipolar cells and CtBP2-tdTomato on graft photoreceptors. In the L7-GFP retina, GFP-positive bipolar cells mostly overlapped with protein kinase Cα (PKCα)-positive bipolar cells, but in L7-GFP/rd, GFP expression was reduced in variable degree in PKCα-positive cells in some parts of the retina (Figures S2B and S2C). GFP-positive host bipolar cells sometimes extend their dendrites even through the remaining graft INL to reach tdTomato-positive graft CtBP2 (Figure 2A ). Although it was sometimes difficult to distinguish graft and host terminals of either bipolar cells or photoreceptors, our labeling approach offers evidence of direct contact between the host L7-GFP-positive bipolar cell dendrites and tdTomato-positive graft photoreceptor synaptic terminals, such as seen between bipolar dendrites and CtBP2 in L7-GFP wild-type retina (Figures 2B and 2C–2C‴). We also stained one of the postsynaptic markers, CACNA1s, that was reported to localize at postsynaptic ribbon synapses and recently identified to cross-react with GRP179 (Hasan et al., 2016, Specht et al., 2009, Tummala et al., 2014). The presence of CACNA1s was observed at the tips of bipolar cells in a wild-type retina (Figure 2D), and CACNA1s immunoreactivities were also present coupled with graft presynaptic terminal, CtBP2-tdTomato, at the tips of L7-GFP-positive bipolar cells, indicating the presence of host-graft synaptic formation (Figures 2E and 2E′). We also observed dendrite tips of PKCα-positive/GFP-negative bipolar cells in the host retina that were in contact with graft regions labeled by CtBP2-tdTomato (Figure S2D), implying that host-graft contact occurred more frequently than was reported by our labels.
Since in the grafts the rosettes were formed with inner/outer segments inward and outer plexiform layer side outward, basically in the correct position to form host-graft interaction, we roughly measured the percentages of the rosette area that can possibly contact host INL (the presence of graft ONL approximately within 10 μm from host retinal margin) (Figure S2E). We estimated that graft photoreceptors of approximately 50.8% ± 7.8% (mean ± SEM, n = 5) of the total graft area may have access to host retina to form synapses.
Light-Responsive Behavior Analyzed by Shuttle-Avoidance System
Because we could not obtain convincing results by optokinetic analysis, on the assumption that, with a small piece of graft in the whole retinal area, these mice may see a very small, spot-like light in some part of their visual field at their best, we adapted a shuttle-avoidance system (SAS) (Figures 3A–3C ; see also Experimental Procedures for detailed protocols) to test visual function. To detect both rod and cone function to the greatest extent possible, we used a mesopic light stimulus of 0.3 cd/m2 with dark-adapted mice, and supplied the mice with 9-cis retinol acetate in case isomerization of all-trans retinol may be inhibited, since the grafted ONLs often form rosettes with their outer segments being separated from the retinal pigment epithelium (RPE). The avoidance ratio is often used as an index (Jiang et al., 1996), but mice tend to move more randomly when they are not confident of avoiding the stimuli, which can increase the avoidance ratio simply by chance. Thus, instead of simply accepting the avoidance rate, we included inter-trial interval (ITI) counts as an additional observation: for every tested animal, we fitted the observed results to a model with three parameters, namely β1, β2, and β3, and estimated the parameters as features of the animal (Figure S3A). Among these parameters, β3 showed the effect of interference with visual function. When the 95% credible interval of the distribution of the estimated β3 was above zero, we judged that interference (mostly transplantation in our study) improved visual function. We included non-treated rd1(rd1/B6) mice as reference data (n = 11) for comparison with each mouse (Figure 3D, black dots and curves). Another rd1 strain, rd1-2J, was also tested on this system to check the reliability of the SAS test, and because rd1-2J were also used in multiple-electrode array (MEA) analysis. When mice were given simultaneous light and audio (beep) signals, the wild-type and rd1-2J mice had similarly high success ratios and low ITIs (Figures S3B and S3C). However, when we switched to a light-only signal, the wild-type mice quickly learned to respond whereas rd1-2J mice did not, with or without 9-cis retinol acetate treatment, much like rd1/B6 mice (Figures 3D and 3E). We then tested the rd1 mice with good iPSC-retina transplants by OCT (Figure S3D). After retinal transplantation, 4 of 10 mice with transplants in both eyes (B) and 5 of 11 mice with a transplant in only one eye (M) were judged to have responded to the light signal (Figures 3F and 3G) while none of the eyes with poor grafting (graft leakage in the vitreous space or retinal detachment in rd1/B6) responded to light. Typical data for a light-responsive grafted mouse and for one with no response are shown in Figure 3H; the light-signal tests are shown in Movies S1 and S2 (for M10 and M8, respectively).
mERG and RGC Recordings from Transplanted Host rd1 Retinas
Next, we used a multielectrode arrays (MEA) system to analyze mERG and RGC recordings. We previously found that mERG in the wild-type retina shows the a- and b-wave-like component similar to the full-field ERG, and the mERG responses were well correlated with responses by RGCs (Fujii et al., 2016). In the wild-type retina, the b-wave that typically represents ON bipolar cell response as well as RGC ON response disappears by the use of mGluR6 receptor agonist L-AP4, whereas in rd1-2J mice, mERG responses were detected only up to 5 weeks old and all the wave components were abolished by L-AP4, indicating the modified intra-retinal signal transmission in these mice (Fujii et al., 2016). In the present study, we transplanted iPSC retinas into rd1 mice at 7 or more weeks of age, and tested their responses by MEA 1.5–4 months later. We analyzed seven retinas after transplantation. The host mouse lines, ages, graft-source iPSC lines and clones, and time after transplantation are summarized in Table 1, along with the number of electrodes on the grafted area and the recorded mERG or RGC responses. The number of RGC responses was objectively calculated followed by the spike sorting and clustering of spike patterns as described in Experimental Procedures and Figures S5A and S5B. We detected mERGs only from the grafted area in all of the samples tested, and representative data are shown in Figures 4A and S4A. The a- and b-wave amplitudes were generally much smaller than those of wild-type retina and grafted retina sometimes elicited irregular mERG wave patterns, but the positive wave (b-wave) was more frequently observed compared with those of young rd1-2J retina (Fujii et al., 2016). The pharmacologic features of mERG and RGC recordings on the representative channels are also shown from the samples TP-5 (Figures 4A–4D ), TP-4, and TP-6 (Figure S4B). Typical a- and b-waves were recorded on channels 15 and 16 (thick graft area), whereas only b-waves were recorded on channels 25 and 26 (graft margins) (Figure 4A). L-AP4 treatment abolished the b-waves (black arrows in Figures 4C and S4B) in both cases, in association with the elimination of transient ON RGC responses (blue arrows in Figures 4D and S4B), similar to the responses in wild-type retina in mERG (Figure 4E), indicating that light-responsive signals were transmitted to ON bipolar cells and then to host RGCs in the grafted area.
| Table 1Summary of Each Retina Sample Used for MEA Analysis | ||||||||||
| Sample | Host | Host Age (Weeks) | Graft Cell Line | Graft Age | Post-TP (Months) | No. of Electrodes | No. of Spike Sources (RGCs) | |||
| On the Graft | With mERG | With RGC Responses | ||||||||
| TP-1 | rd1/B6 | 8 | Nrl-GFP | DD14 | 3 | 19 | 4 | 3 | 5 | |
| TP-2 | rd1-2J | 9 | Nrl-GFP | DD14 | 4 | 15 | 8 | 5 | 6 | |
| TP-3 | rd1-2J | 7 | Nrl-GFP | DD13 | 2 | 9 | 8 | 1 | 1 | |
| TP-4 | L7-GFP/rd1 | 8 | Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato#1 | DD13 | 1.5 | 15 | 12 | 3 | 8 | |
| TP-5 | L7-GFP/rd1 | 8 | Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato#2 | DD13 | 2 | 43 | 58 | 49 | 112 | |
| TP-6 | L7-GFP/rd1 | 8 | Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato#2 | DD13 | 2 | 51 | 44 | 10 | 17 | |
| TP-7 | L7-GFP/rd1 | 8 | Nrl-GFP/ROSA::Nrl-CtBP2-tdTomato#3 | DD13 | 2 | 8 | 6 | 0 | 0 | |
The following information was provided for each tested retina. The host age/graft age in differentiation days (DD) at the time of transplant; the number of months of post-transplant (TP) MEA recordings; the number of electrodes present; the number of electrodes on the grafted area, those positive for mERG recordings, and those positive for RGC responses; and the number of spike sources (RGCs) that were categorized in a cluster other than the non-specific pattern.
Clustering RGC Light Responses in the Host rd1 Retina after Transplantation
We also adapted deep-learning methods to cluster the RGC spike patterns into groups that included both ON and OFF responses (Figures 5A and 5B ). Transient ON, OFF, and ON-OFF responses were most dominant in the wild-type (B6) retina with a full-field light flash of 0.45 log cd/m2; these shifted to a transient OFF response when mGluR6 was blocked (Figure 5A). In retinas from 7-week-old or older rd1-2J mice, all of the spikes were clustered as non-specific patterns except for 0.2% of the ON-hyperactive pattern. However, we detected transient ON responses consistently in the grafted area, with an average of 5% (0% to 16% at maximum) of all the detected RGC sources from the whole recorded area (Figure 5A). The number of detected response sources (RGCs) and their patterns are shown for each tested retina in Figure 5B. Generally the grafts that covered a larger number of electrodes elicited a higher number of RGC responses, whereas in the samples where grafts were placed on a small number of electrodes in the experimental setting (TP-3 and TP-7, with 9 and 8 electrodes, respectively) only few or no typical RGC responses were recorded (Table 1). When the number of typical light responses (transient ON/transient OFF/ON-OFF) was plotted against the number of electrodes with graft, it showed a linear correlation tendency except for TP-5, which showed an exceptionally good result among the tested samples (Figure 5C). These RGC responses over the graft were clearly distinguishable from residual RGC activity in the degenerating rd1 retina; rd1 retina elicited either a non-specific pattern with regular intensity stimuli, or hyperactive patterns (ON, OFF, and delayed) shown as clustering groups mostly with higher-intensity stimuli (3.01 log cd/m2) as in the training dataset (Figure S5A).
3D Image Reconstruction of the Samples after MEA Recordings
To eliminate the possibility of a contaminant response from graft cells, we reconstructed 3D histological images of the MEA-recorded area after MEA analysis and carefully observed host-graft integration over the MEA-recorded channels (Figures 6A–6C ). The sectional views over the channels presented in Figure 4D (channels 16 and 25) revealed that the Nrl-GFP-positive graft ONLs expressed the presynaptic marker CtBP2-tdTomato at the host-graft interfaces (red arrows) in contact with the host INLs (white arrows), which contained L7-GFP-positive bipolar cells (green arrows). Channel 16 showed larger, thicker graft ONL than those observed in channel 25, consistent with the presence of marked a-waves on channel 16 but not channel 25 (Figure 4C).
Discussion
We have previously shown that retinal tissues differentiated from mouse or human ESCs or iPSCs could develop to form ONLs consisting of mature photoreceptors with highly differentiated structures such as inner/outer segments after transplantation in the degenerated host retina (Assawachananont et al., 2014, Shirai et al., 2015). We also showed that these graft ONL integrated as a structured layer to the host inner layers possibly with host-graft synaptogenesis. However, the functional evaluation was yet to be performed.
In the current study, with the use of Nrl-GFP/CtBP2-tdTomato cell lines with host L7-GFP/rd1 mice, we readily visualized direct contact between the host-graft cells, and our observations revealed that host bipolar cells extend their dendrites into the graft, sometimes even through the remaining graft inner cells (Figure 1C). Retraction of bipolar cell dendrites were observed after photoreceptor degeneration (Marc et al., 2003), and transplanted photoreceptors seem to provide some environmental change for the host bipolar cells to regrow dendrites. The reason for and effect of the variable degree of host L7-GFP expression in PKCα-positive rod bipolar cells with rd1 phenotype is not known, but the use of the present host-graft combination may further help to quantitatively access the host-graft integration in our future studies in optimizing the transplantation conditions.
To evaluate visual function through behavioral tests, we first tried optokinetic testing (OKT). Some of the mice with retinal transplants did not noticeably track the moving bars but became exceptionally static. Considering the relatively small graft area and the unknown efficiency of synaptogenesis, these mice probably saw a very small, spot-like light in some part of their visual field. We concluded that OKT was inadequate for detecting visual function in these mice. In addition, OKT has shown conflicting results in retinal degeneration and should be used with caution (McGill et al., 2012). Likewise, these transplanted mice will not follow regular light-avoidance behavior patterns if they only see small or ambient light. This led us to adapt SAS for objective evaluation of their behavior patterns in response to light. The SAS requires approximately 2 weeks of training with beep and light, and 2 more weeks of evaluation with a light-only signal, so we restricted the use of analysis to only the mice with a substantial amount of subretinal graft as judged by in vivo OCT imaging after transplantation. There was no difference in the results between mice with transplants in one or both eyes, and we assume that the graft area, and possibly the location, may contribute to the sensitivity of visual perception in our SAS evaluation.
For the photoreceptors to function, recycling of visual pigments by isomerization and oxidation, from all-trans retinol to 11-cis retinol, and to 11-cis retinal, is essential (Parker and Crouch, 2010). The first essential step of isomerization to form 11-cisretinol is catalyzed by RPE65 in RPE for rod visual pigments (Redmond et al., 1998) and in Muller cells for cone visual pigments (Travis et al., 2005). The further oxidation to restore 11-cis retinal is then catalyzed by retinol dehydrogenases (RDHs), which are distributed in retina and RPE in a number of isoforms with overlapping activities (Maeda et al., 2007, Parker and Crouch, 2010). Because the grafted ONLs often form rosettes with their outer segments being separated from the RPE, isomerization of all-trans retinol may be inhibited. Since the treatment of 9-cisretinyl acetate (9-cis retinol acetate) restored the impaired visual function in Rpe65−/− mice (Maeda et al., 2009), we supplied 9-cis retinol acetate when we performed a behavior test with light-only signals to supply a substantial amount of the source of 11-cis retinal, although not the direct competent form. Partly because SAS was a time-consuming test, we did not perform the experiments with or without the drug treatment, or the tests after washout, so we were unable to determine the effects of 9-cis retinol acetate supplementation. Treatment of 9-cis retinol acetate had no effect on unsuccessful transplantation or non-treated rd1 mice, so the change in SAS results after transplantation is not due to the protective effect by the treatment.
In electrophysiological evaluation using MEA, we carefully evaluated the graft-originated responses not simply by their presence or absence but by their functional properties. The presence of mERG responses were hardly detected in rd1 host retinas at 7 weeks or older, and, even in earlier degeneration, all the wave components were eliminated by mGluR6 blockade by L-AP4 (Fujii et al., 2016). After transplantation, marked light-responsive mERGs were recorded in the grafted area in all the samples tested, although the amplitudes were smaller than those of wild-type retina and the wave patterns were more variable, indicating that graft is responsive to light. The presence of the remaining negative-wave components after mGluR6 blockade implies that these responses may originate either from the graft photoreceptors or OFF components of host second neurons, which were both absent in rd1 host retinas before transplantation (Fujii et al., 2016). Furthermore, we were able to record significant light-responsive spikes from the host RGC layer, most of which were clustered as transient ON patterns. With our MEA system, approximately 20 cells are on one electrode in the RGC layer, half of which are reportedly amacrine cells (Jeon et al., 1998). Possible flaws in interpreting these RGC recordings include (1) residual responses from the host (cone) photoreceptors, (2) light-dependent RGC responses from intrinsically photosensitive retinal ganglion cells (iRGCs) (Berson et al., 2002, Hattar et al., 2002, Pickard and Sollars, 2012), and (3) spikes from graft cells, including graft RGCs, that may have been exposed on the host RGC surface layer. As we have described, the possibility of residual host transient ON function is unlikely. The possibility of iRGC activities can also be discounted, because the L-AP4 blockage of mGluR6 demonstrated that the RGC responses were postsynaptic. We also observed that in the area where light-responsive RGCs were recorded, the RGC and inner layers of host retina was present by histological examination, leaving little possibility that these RGC responses were mostly contributed by exposed graft cells. Interestingly, the channels that elicit RGC responses and those with evident mERG responses are not completely the same, such as seen in TP-6 in Figure S4A, which indicated that the former may represent the graft status such as the presence of graft photoreceptor outer segments in correct orientation, whereas the latter may represent the location of RGCs that eventually received input from graft light response. A few RGC responses detected outside the graft margin, as shown in Figure 4B, may also support that the responses derive from the host RGCs that may include the graft area in their receptive fields.
This proof-of-concept study showed that the iPSC retina, when transplanted into the eyes of end-stage retinal-degeneration mice, develops a mature ONL and responds to light. Nearly half of the mice with retinal transplant showed light-responsive behavior. We recently showed that the hESC retina can develop and mature after being transplanted into the eyes of monkeys with ONL-lost retinal degeneration (Shirai et al., 2015). We are currently testing the competency of a human iPSC retina to restore visual function.