Visualizing functional retinal integration of transplanted retinal ganglion cells

移植视网膜神经节细胞的功能性视网膜整合可视化

• 批准号: 10707349
• 负责人: Thomas Vincent Johnson
• 金额: $ 20.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10707349
• 关键词: 3-Dimensional; Achievement; Address; Animal Model; Blindness; Brain; Cell Communication; Cell Death; Cell Differentiation process; Cell Line; Cell Separation; Cell Survival; Cell Transplantation; Cells; Cellular Structures; Central Nervous System; Chimeric Proteins; Chronic; Color; Confocal Microscopy; Coupled; Custom; Dendrites; Detection; Development; Devices; Discrimination; Disease; Disease model; Electrophysiology (science); Engraftment; Enterobacteria phage P1 Cre recombinase; Event; Eye; Fluorescence; Foundations; Future; Gene Expression; Gene Transfer Techniques; Generations; Genetic Recombination; Genomics; Glaucoma; Green Fluorescent Proteins; Heterogeneity; Human; Image; Imaging Device; Injury; Inner Plexiform Layer; Kinetics; Label; Lasers; Light; Membrane; Methods; Microscopy; Molecular; Mus; Nervous System; Neurons; Neuropathy; Ophthalmoscopy; Optic Nerve; Optics; Pathogenesis; Pathway interactions; Patients; Persons; Phenotype; Pluripotent Stem Cells; Production; Recombinant adeno-associated virus (rAAV); Regenerative Medicine; Reporter; Reporting; Resolution; Retina; Retinal Ganglion Cells; Retrieval; Rodent; Scanning; Specificity; Speed; Stem cell transplant; Structure; Synapses; System; Techniques; Therapeutic; Time; Tracer; Transgenic Organisms; Transplantation; Visual Pathways; Visualization; Wheat Germ Agglutinins; Work; adaptive optics; adaptive optics scanning laser ophthalmoscopy; adeno-associated viral vector; axon regeneration; blind; cell replacement therapy; clinical translation; cost effectiveness; electrical measurement; flexibility; fluorophore; high throughput screening; human stem cells; improved; in vivo; in vivo engraftment; in vivo imaging; innovation; instrumentation; lens; migration; multimodality; neural circuit; nonhuman primate; novel; novel strategies; optic nerve disorder; patch clamp; post-transplant; postsynaptic; presynaptic; red fluorescent protein; response; retina transplantation; retinal neuron; serial imaging; sight restoration; synaptogenesis; time use; tool; transcriptomics; transplantation therapy

PROJECT SUMMARY Functional retinal ganglion cell (RGC) replacement could restore vision to tens of millions of people who are blind from glaucoma and other optic neuropathies. Establishment of techniques for RGC differentiation from pluripotent stem cells and the achievement of long-distance endogenous axon regeneration within the optic nerve support the promise of RGC replacement therapies. However, clinical translation requires significant improvements in the functional integration of transplanted RGCs into the host retinal neurocircuitry. To enable the study of synaptogenesis by transplanted neurons, we propose developing and validating an innovative, versatile, sensitive, experimental tool that leverages transsynaptic transport of wheat germ agglutinin (WGA) protein fused to Cre recombinase to enable expression of a Cre-dependent reporter in synaptically integrated donor neurons. The label is durable, facilitating downstream applications such as single cell isolation and transcriptomic analysis, and bidirectional to allow the labeling of either pre- or post-synaptic graft-host neuronal partners. Here, we propose to establish human pluripotent cell lines to be used in conjunction with highly efficient recombinant AAV vectors to report the functional retinal of transplanted RGCs. Since expression of the fluorescent reporter is automatic upon genomic recombination, the tool will facilitate real-time detection of functional neuronal integration in vivo. We propose a short wavelength (blue) fluorescent reporter for synaptogenesis, which will be compatible with additional red and green fluorescence for multicolor microscopy and ophthalmoscopy. We will characterize the kinetics of expression and validate the specificity of this synaptogenesis reporter tool using a combination of high-resolution confocal microscopy, immunolabeling of synaptic machinery, and single-cell electrophysiology within retinal flatmounts. Further, we will study the functional integration of transplanted human RGCs in vivo using a custom-built multicolor adaptive optics scanning laser ophthalmoscope. The instrumentation provides subcellular resolution within in the xy plane and depth discrimination with the retina through z-stacking. Longitudinal imaging of living eyes post- transplantation will therefore enable the correlation of donor RGC dendrites targeting of the inner plexiform layer with reporting of functional synaptogenesis. We will characterize the structural events leading to retinal integration of donor RGCs. This work will provide a foundation for future experimentation that includes manipulation of the host microenvironment to improve engraftment efficiency, in vivo optical electrophysiology of integrated RGCs, and robust analyses of RGC interactions with various resident retinal cells using cell-specific reporter mice.

项目概要 功能性视网膜神经节细胞（RGC）替代可以使数千万人恢复视力 因青光眼和其他视神经病变而失明的人。技术的确立 用于从多能干细胞分化出 RGC 并实现远距离 视神经内的内源性轴突再生支持 RGC 替代的前景 疗法。然而，临床转化需要功能上的显着改进 将移植的 RGC 整合到宿主视网膜神经回路中。为了使研究能够 通过移植的神经元进行突触发生，我们建议开发和验证一种创新的、 利用小麦胚芽的跨突触运输的多功能、灵敏的实验工具 凝集素 (WGA) 蛋白与 Cre 重组酶融合，以实现 Cre 依赖性蛋白的表达 突触整合供体神经元中的报告者。标签耐用，有利于下游 单细胞分离和转录组分析等应用，以及双向允许 突触前或突触后移植宿主神经元伙伴的标记。在此，我们建议 建立与高效重组细胞结合使用的人类多能细胞系 AAV 载体报告移植 RGC 的功能性视网膜。由于表达了 荧光报告基因在基因组重组时自动进行，该工具将有助于实时 体内功能性神经元整合的检测。我们建议使用短波长（蓝色） 用于突触发生的荧光报告基因，与额外的红色和绿色兼容 用于多色显微镜和检眼镜检查的荧光。我们将描述其动力学 表达并使用组合验证该突触发生报告工具的特异性 高分辨率共聚焦显微镜、突触机制的免疫标记和单细胞 视网膜平片内的电生理学。进一步，我们将研究功能集成 使用定制的多色自适应光学扫描激光器在体内移植人类 RGC 检眼镜。该仪器提供 xy 平面内的亚细胞分辨率 通过 z 轴堆叠进行视网膜深度辨别。活体眼睛术后纵向成像 因此，移植将使供体 RGC 树突靶向的相关性成为可能。 内丛状层具有功能性突触发生的报告。我们将表征 导致供体 RGC 视网膜整合的结构事件。这项工作将提供一个 为未来的实验奠定基础，包括操纵宿主微环境 提高植入效率、集成 RGC 的体内光学电生理学以及鲁棒性 使用细胞特异性报告小鼠分析 RGC 与各种驻留视网膜细胞的相互作用。