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

VRC：基于干细胞的激光引起永久性视网膜损伤的治疗策略

• 批准号: 10206151
• 负责人: Biju Blavady Thomas
• 金额: $ 23.69万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206151
• 关键词: Ablation; Age; Anesthesia procedures; Animal Model; Animals; Architecture; Behavior; Behavioral; Blindness; Bruch' s basal membrane structure; Cell Therapy; Cell Transplantation; Cells; Choroid; Data; Deterioration; Devices; Discrimination; Disease; Electrophysiology (science); Electroretinography; Euthanasia; Exposure to; Eye; Eye Injuries; Graft Rejection; Harvest; Human; Immunohistochemistry; Immunosuppression; Implant; Injury; Investigation; Laboratories; Laser injury; Lasers; Lead; Low-Level Laser Therapy; Maps; Masks; Membrane; Methods; Military Personnel; Modeling; Morphology; Mydriasis; Natural regeneration; Nude Rats; Operative Surgical Procedures; Optical Coherence Tomography; Organoids; Oryctolagus cuniculus; Pathologic; Pharmaceutical Preparations; Photoreceptors; Phototransduction; Pigments; Rattus; Recovery of Function; Research Personnel; Retina; Source; Structure; Structure of retinal pigment epithelium; Surface; Synapses; Techniques; Testing; Tissue Engineering; Tissues; Transplantation; Transplantation Surgery; United States National Institutes of Health; Vision; Visual; base; behavior test; cell behavior; cell replacement therapy; effective therapy; experimental group; experimental study; human embryonic stem cell; human embryonic stem cell transplantation; implantation; improved; monolayer; parylene; post-transplant; prevent; reconstruction; repaired; response; retinal damage; retinal imaging; retinal progenitor cell; stem cell derived tissues; stem cell therapy; stem cells; superior colliculus Corpora quadrigemina; treatment strategy

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.

在战场上，眼睛的伤害可以由各种来源造成。激光装置广泛用于武装部队 可导致光感受器（PR）的不可逆损失和脉络膜损伤，导致永久性视觉障碍。 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。细胞替代 治疗是对激光永久性损伤的视网膜进行结构和功能重建的主要希望。 这项研究的目的是通过使用“全视网膜贴片”来开发激光眼损伤的细胞疗法 由健康的RPE、人造布鲁赫膜（BM）和视网膜类器官（RO）片制成。我们的假设 永久性视网膜损伤可以通过使用这种干细胞衍生的组织来修复（并改善视力）， 工程共移植。将RO片和极化RPE片在人工晶状体上培养制成的组合移植物， BM会对严重受损的视网膜进行结构重建，失去的视力会得到更多的改善 与单独的RPE或单独的视网膜类器官的移植相比，使用共移植物。由于RPE单层是 对于维持健康的PR是不可或缺的，并且在光转导循环中与PR相互作用，我们认为， 健康极化RPE单层的存在和来自完整和健康BM的结构支持将提供 有利于再生RO膜的微环境。此外，聚对二甲苯膜可以保护聚对二甲苯膜。 从患病宿主眼睛的病理BM表面共移植，以防止BM异常不利地 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中得到验证 代表军事相关损伤的模型：（1）视网膜激光损伤的大鼠模型。(2)兔激光损伤 眼睛大小更接近人类的模型。NIH裸大鼠和正常荷兰带兔将 在麻醉和瞳孔放大后接受激光照射。视力丧失将通过视网膜电图（ERG）确认 和行为测试。动物将接受联合移植植入物或对照手术。愿景 将通过视网膜电图（ERG）确认损失。以下实验组将用于2个目标： (a)人胚胎干细胞（hESC）-RO+RPE+BM的移植片;骨髓匹配的对照包括： (b)仅RO片;（c）仅RPE片;（d）无手术。免疫缺陷大鼠将用于所有实验， 避免移植物排斥反应，而无免疫抑制药物的负面影响。移植位置将是 通过光学相干断层扫描（OCT）验证。大鼠的视力将每月通过视动/视觉测试进行测试。 辨别测试和ERG。在实验结束时（移植后3-6个月），视觉反应 将通过电生理学映射到上级丘。研究人员将对老鼠的 实验条件术后2个月对家兔实施安乐死。将分析大鼠和兔视网膜 通过免疫组织化学检测供体和宿主突触标记物以及视网膜细胞标记物的存在，以确认 移植物成熟并与宿主视网膜整合。