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  • We experienced difficulties in observing maturation of hESC


    We experienced difficulties in observing maturation of hESC retinas in rd1 mice even with immunosuppression, but with NOG-rd1-2J mice the transplanted hESC retinas consistently developed mature ONL with rod and cone opsins. Furthermore, graft photoreceptors show signs of developing IS/OS structures as evidenced by electron microscopy. However, we had not yet shown any function by hESC/iPSC retina following transplantation. In the present study, light-responsive RGC activities were detected in 3 of 8 retinas over the grafts by MEA recording from 28-week-old or older NOG-rd1-2J mice (20 weeks or more after transplantation). At such advanced stage of retinal degeneration or even younger NOG-rd1-2J, no remaining cone activity was observed in the proximal retina where the grafts usually lay. Although we could not completely deny the rescue effect on the host’s remaining cones by grafts, some of the light responses recorded after transplantation were clearly distinguished by their proximity to the disc and location directly over the graft. These responses were not sparsely covering only a few electrodes as in the non-transplanted retina but were detected in clusters covering multiple electrodes directly in the grafted area, suggesting that they are more likely driven by the graft photoreceptors rather than remaining cones. Hypersensitive response patterns were initially observed in the degenerating retinas, preferentially to a bright light stimulus but barely with our regular light stimuli at 0.45 log cd/m2/s (Mandai et al., 2017). After transplantation, however, many RGCs, usually located in and around the grafted area, were classified as “light-responsive hypersensitive” even to the regular light stimuli. Some of the hypersensitive responses were rather ambiguous as they cannot be clearly distinguished from the normal patterns, such as whether to be sustained ON or ON-hyperactive. We propose that these patterns represent either degenerating or regenerating processes and may possibly originate from the immature host-graft synaptic connections or tegison synthesis associated with the circuitry formation. No OFF responses were detected upon L-AP4 block, suggesting that the OFF pathway may not yet have been recovered. The L-AP4-insensitive hypersensitive activities may be partly derived from intrinsically photosensitive RGCs, despite the use of Opsinamide. Alternatively, these responses may suggest a wrongly or immaturely rewired circuitry tethered to the rather excitable RGCs in degenerating retina (Euler and Schubert, 2015). It is noteworthy that we obtained light responses in one sample even before the 9-cis-retinal treatment (Figure 6B), which indicates that graft photoreceptors were readily functional with functional visual pigments in vivo, even though the rosette formation dampens normal contact with RPE cells. In the present study, we did not detect full-field ERG response after transplantation. Since detectable ERG response is estimated to require more than 150,000-cell integration (Pearson et al., 2012), our host-graft integration is not considered to be sufficient. However, considering that a patient with non-recordable full-field ERG still has a limited visual field, the absence of in vivo full-field ERG does not indicate total blindness. For the ex vivo recording, although the evident mERGs were elicited in miPSC retina grafts at 5 weeks after transplantation, we did not detect mERG in hESC retina grafts to the same 10-ms flashlight (1/100 photons compared with the multiunit recordings of RGC). Since the mERG responses in miPSC were mostly abolished by L-AP4, the wave components likely represented the postsynaptic bipolar cells and RGCs of either graft or host rather than the properly oriented photoreceptors of limited amount inside the graft rossettes (Fujii et al., 2016). The lack of mERG wave components but not RGC multiunit responses in hESC retina suggests that the graft photoreceptors did not convey a strong enough response to the weaker stimulus. Still, we obtained RGC responses in 3 of 7 retinas with a substantial hESC transplant, which suggests the presence of functional hESC retinal photoreceptors, although RGC responses were detected in all samples from miPSC retina-transplanted NOG-rd1-2J mice. The lower responsiveness of hESC retina compared with miPSC retina in NOG-rd1-2J mice may be attributed to (1) fewer functional photoreceptors in hESC retina than in miPSC retina, (2) the advanced and aged host environment at the time of synaptic integration of hESC retinas, since hESC retina takes a longer time to reach synaptogenesis stage than does miPSC retina, and (3) inefficient synaptic formation in xenotransplantation compared with allotransplantation as suggested by Laver and Matsubara (2017). However, this incompatibility of hESC/iPSC retina would not be an issue in human host retinas. Finally, (4) the Crx::venus ESC graft we have used here may have negatively affected synaptogenesis, since functional maturation is reportedly delayed in Crx+/− mice (Furukawa et al., 1999).