![]() ![]() ![]() In the present study, therefore, we conducted hiPSC-retinas transplantation about 2 months after the laser photocoagulation (62 days for the cynomolgus monkey and 53 days for the rhesus monkey). The retinas were detached from the electrodes after recording and fixed with 4% paraformaldehyde (PFA) at room temperature for immunostaining and confocal imaging. Recordings to the same set of light stimuli were repeated before, during and after the L-AP4 application (10 μm 016–22083, Wako), for which blocks the metabotropic glutamate receptor type 6 (mGluR6)-mediated photoreceptor-ON bipolar cell transmission, to confirm the photoreceptor-origin of detected light responses. We categorized the detected RGCs to spontaneous firing, ON/OFF/ON-OFF/Delayed-ON light response types, or hypersensitive type automatically, based on their temporal spiking pattern corresponding to the light stimulus onset and offset using the homemade R scripts. Field potentials were recorded for a 10-s duration to reveal the micro-ERG (mERG) b-waves to 10-ms stimuli RGC action potentials to 1-s light stimuli were recorded for a 21-s duration and sorted offline (Spike2, Version 7.20, CED) to separate individual cells with spikes. IR-DIC images of retinas mounted on electrodes were taken during and after recording to determine the correlation of electrodes and grafts with Recoverin and/or STEM121 immunoreactivity afterwards. Note that since the transplanted hiPSC-retinal sheets were not engineered for any fluorescence labeling, the grafted areas were targeted by identifying the whitish spotted regions in between the optic nerve disc and the entry holes made during transplantation. After removing the vitreous body, the freshly isolated retina was trimmed to a degree that both optic nerve disc and mid-peripheral regions were retained, including the potentially grafted areas, before mounting on the electrodes (MED-P515A for MED64 system 60MEA200/30iR-Ti-gr for MEA60 system) with the retinal ganglion cell (RGC) side down. The eyecups were carefully washed and incubated with constantly oxygenated Ames' medium (A1420, Sigma-Aldrich) in the dark at room temperature before use. In brief, under dim red light peaked at 690 nm, the animals were deeply anesthetized by intramuscular injection of the ketamine-xylazine mixture, and euthanatized by sevoflurane inhalation immediately after enucleation. Since the use of iPS cells would allow for the application of cell therapy by overcoming the ethical/religious issues as well as for the use of varieties of choices of cell lines such as HLA-matched iPSC cell lines, the examinations for competency of iPSC-retina as graft source was of great importance in view of clinical application. These studies imply the competency of humen ESC-derived retinas but also leave the difficulties in obtaining functional data from xeno-transplantation of neural tissues/cells where reconstruction of host-graft neural network is required. We also reported that xeno-transplanted human ESC-derived retinas mature in the immunodeficient end-stage retinal degeneration models of rodents and monkeys, and can recover light responses in a mouse model but of less robustness compared to allo-transplantation. We have previously reported that allo-transplantaion of mouse ES/iPSC-retinas can develop structured photoreceptor layer after transplantation and drive light responses in the end-stage retinal degeneration retinas with few remaining photoreceptors. Possible restoration of visual function in the end-stage retinal degeneration rodent models by rodent and human fetus retinas and ES/iPSC-derived retinal tissues/cells have been suggested in past years. The Lancet Regional Health – Western Pacific.The Lancet Regional Health – Southeast Asia.The Lancet Gastroenterology & Hepatology. ![]()
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