Retinal pigment epithelium (RPE) and photoreceptors depend on each other for normal function and survival. Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) lead to irreversible loss of vision in millions worldwide, due to RPE dysfunction and photoreceptor degeneration. Our long-term goal is to reverse vision loss by a combined transplant of neural retina sheets containing photoreceptor progenitors, plus a polarized functional RPE monolayer to support the photoreceptors. Human embryonic stem cells (hESCs) differentiated into RPE and retina organoids (RO) - which would substitute for neural retina sheets - can provide an unlimited supply of clinically applicable retinal donor tissue. Retinal sheet transplants - dissected from hESC-ROs – develop photoreceptors, improve visual acuity, responses to light, and integrate with the host retina. However, RO’s are generally devoid of a polarized RPE layer. Therefore, cografting of hESC–derived neural retina and a healthy RPE monolayer is needed for treatment of advanced disease conditions; but have not been performed on a large scale. To advance cell replacement therapy, the mechanism of action of the transplant should be well understood. Although synaptic connectivity of retinal transplants has been ascribed a role for visual improvement, details regarding the circuitry between transplant and host retina remain unknown. To obtain better insight, retinal degenerate (RD) rats that express a specific label to identify defined host retinal neuron populations need to be used. Thus, the goal of this project is to improve the efficiency of transplants and analyze the functional recovery in relationship to neural connectivity. The proposed studies are aimed (1): To determine the efficacy of cografting “complete transplants”. We hypothesize that “complete” transplants consisting of hESC-neural retina together with RPE will improve visual recovery - compared to transplanting hESC-neural retina or RPE alone. Royal College of Surgeons (RCS) rats, a model of a hereditary (chronic) RPE defect, at an advanced stage of RD will receive either “cografts”, or transplants of ROs or RPE alone, and analyzed long-term (6-8 months) after grafting. The successful outcome of this Aim should allow us to quantitatively evaluate the improved outcome of cografting. (2): To determine mechanisms of functional recovery by detailing transplant-host connectivity. We hypothesize that visual improvement will correlate with integration and synaptic connectivity of neural retinal sheet transplants in the host retina. This will be tested by labels for both the donor and specific retinal neurons of the host retina. In collaboration with Envigo, we will produce immunodeficient rhodopsin mutant (Rho S334ter-3) RD recipients expressing Td-Tomato either universally (cre/lox inducible) or in CaMKII retinal neurons (using CRISPR-Cas9 technology), which will allow us to examine in detail the connectivity of RO transplants with spec...