VRC: A Stem Cell-Based Treatment Strategy for Laser-Induced Permanent Retinal Damages

点击翻译按钮获取中文摘要

Eye damage can be caused by various sources in a battlefield. Laser devices widely used by the armed forces can lead to irreversible loss of photoreceptors (PRs) and damage to the choroid, leading to permanent visual loss. Currently there are no effective treatments for laser-induced permanent retinal damage. Cell replacement therapy is a major hope for structural and functional reconstruction of a retina permanently damaged by laser. The proposed study's objective is to develop a cellular therapy for laser eye injuries by using a “total retina patch” made of healthy RPE, an artificial Bruch's membrane (BM), and sheets of retina organoids (RO). Our hypothesis is that permanent retinal damage can be repaired (and vision improved) by using this stem-cell-derived tissue- engineered co-graft. A combination graft made of RO sheets and polarized RPE sheets cultured over an artificial BM will produce a structural reconstruction of a severely damaged retina, and lost vision will be more improved using a co-graft than a transplant of either RPE alone or retina organoid alone. Since the RPE monolayer is integral to maintaining healthy PRs, and interacts with the PRs in the phototransduction cycle, we think that the presence of a healthy polarized RPE monolayer and structural support from intact and healthy BM will provide a beneficial microenvironment for regenerating the RO sheets. Further, the parylene membrane can protect the co-graft from the pathological BM surface of the diseased host eye to prevent BM abnormalities from unfavorably altering the transplanted cells' behavior. This hypothesis will be tested in two different laser damage animal models that represent military-relevant injuries: (1) Rat model for retinal laser damage. (2) Rabbit laser damage model having eye size more comparable to that of humans. NIH nude rats and Normal Dutch Belted rabbits will be exposed to laser after anesthesia and pupil dilation. Vision loss will be confirmed by electroretinogram (ERG) and behavioral testing after laser exposure. The animals will receive co-graft implants or control surgeries. Vision loss will be confirmed by electroretinogram (ERG). The following experimental groups will be used for the 2 Aims: (a) Transplants of sheets of human embryonic stem cell (hESC)-RO+RPE+BM; Age-matched Controls include: (b) RO sheets only; (c) RPE sheets only; (d) no surgery. Immunodeficient rats will be used in all experiments to avoid graft rejection without the negative effects of immunosuppression drugs. Transplant placement will be verified by optical coherence tomography (OCT). The rats' vision will be tested monthly by optokinetic/ visual discrimination testing and ERG. At the end of the experiment (3-6 months post transplantation), visual responses will be mapped in the superior colliculus by electrophysiology. Investigators will be masked to the rats' experimental condition. Rabbits will be euthanized 2 months post-surgery. Rat and rabbit retinas will be analyzed by immunohistochemistry for the presence of donor and host synaptic markers and retinal cell markers to confirm transplant maturation and integration with the host retina.