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

在战场上，眼睛损伤可能由各种来源造成。军队广泛使用的激光设备 可能导致不可逆转的光感受器(PR)丧失和脉络膜损伤，导致永久性视力 损失。目前还没有有效的治疗激光引起的永久性视网膜损伤的方法。电池更换 治疗是激光永久性损伤的视网膜结构和功能重建的主要希望。 这项研究的目的是开发一种使用“全视网膜贴片”治疗激光眼损伤的细胞疗法。 由健康的RPE、人造Bruchs膜(BM)和视网膜有机体(RO)片组成。我们的假设 永久性的视网膜损伤可以通过使用这种干细胞来源的组织来修复(并改善视力)- 精心设计的联合移植。RO片材和极化RPE片材在人工上培养的组合移植物 BM将对严重受损的视网膜进行结构重建，丧失的视力将得到更多改善 使用联合移植比单独移植RPE或单独移植视网膜器质要好。由于RPE单层是 为了维持健康的PR，并在光转导周期中与PR相互作用，我们认为 存在健康的极化RPE单层以及来自完整和健康的BM的结构支撑将提供 有利于反渗透板材再生的微环境。此外，该对苯膜还可以保护 从病眼的病理性BM表面共移植来防止BM异常 改变移植细胞的行为。这一假设将在两种不同的激光损伤动物身上进行验证 代表军事相关损伤的模型：(1)大鼠视网膜激光损伤模型。(2)兔激光损伤 眼睛大小更接近人类的模特。NIH裸鼠和正常荷兰皮带兔将 麻醉、散瞳后暴露于激光照射。视力丧失将通过视网膜电图(ERG)确认 以及激光照射后的行为测试。这些动物将接受联合移植或对照手术。视觉 丢失将通过视网膜电图(ERG)确认。以下试验组将用于这两个目标： (A)移植人类胚胎干细胞(HESC)-RO RPE BM；年龄匹配的对照包括： (B)仅RO床单；(C)仅RPE床单；(D)不做手术。免疫缺陷大鼠将在所有实验中用于 避免移植物排斥反应，而不受免疫抑制药物的负面影响。移植安置将是 经光学相干层析成像(OCT)验证。每月将对大鼠的视力进行视动/视觉测试 辨别试验和ERG。在实验结束时(移植后3-6个月)，视觉反应 将通过电生理学被定位在上丘。调查人员将对老鼠们戴上面具 实验条件。术后2个月处死兔。大鼠和兔的视网膜将被分析 通过免疫组织化学方法检测供体和宿主的突触标志物和视网膜细胞标志物的存在 移植的成熟和与宿主视网膜的整合。

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.