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: 10510837
• 负责人: Thomas Vincent Johnson
• 金额: $ 24.56万
• 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/10510837
• 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; Chimeric Proteins; Chronic; 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; Heterogeneity; Human; Image; Imaging Device; Injury; Inner Plexiform Layer; Kinetics; Label; Lasers; Light; Membrane; Methods; Microscopy; Molecular; Mus; Nervous system structure; Neuraxis; Neurons; Neuropathy; Ophthalmoscopy; Optic Nerve; Optics; Pathway interactions; Patients; Persons; Phenotype; Pluripotent Stem Cells; Production; Proteins; 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; 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.

项目总结 功能性视网膜神经节细胞(RGC)置换可恢复数千万人的视力 因青光眼和其他视神经疾病而失明的人。技术的确立 多能干细胞向RGC分化并实现远距离分化 视神经内源性轴突再生支持RGC替代的前景 治疗。然而，临床翻译需要在功能上有显著的改进 移植的视网膜神经节细胞与宿主视网膜神经回路的整合。使研究成为可能 通过移植神经元的突触发生，我们建议开发和验证一种创新的， 多功能、灵敏的实验工具，利用小麦胚芽的跨突触运输 凝集素(WGA)蛋白与Cre重组酶融合以实现Cre依赖的表达 记者在突触整合的供体神经元。标签经久耐用，便于下游 单细胞分离和转录分析等应用，以及允许 标记突触前或突触后移植物-宿主神经元伙伴。在此，我们建议 建立可与高效重组人联合使用的人多能细胞系 AAV载体报道移植的RGC的视网膜功能。由于表示了 荧光报告在基因组重组时是自动的，该工具将促进实时 体内功能神经元整合的检测。我们建议使用短波长(蓝色) 突触发生的荧光报告，它将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。我们将描述它的动力学 使用组合表达并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，以及单细胞 视网膜平铺内的电生理学。在此基础上，我们将进一步研究功能集成。 自制多色自适应光学扫描激光器体内移植人视网膜神经节细胞 检眼镜。该仪器在xy平面内提供亚细胞分辨率，并且 通过z堆积与视网膜进行深度辨别。术后活眼的纵向成像 因此，移植将使供者RGC树突靶向 内丛状层，有功能性突触发生的报道。我们将描述 导致供体视网膜节细胞视网膜整合的结构性事件。这项工作将提供一种 为未来的实验奠定基础，包括操纵宿主微环境以 提高植入效率、体内集成视网膜节细胞的光学电生理和健壮性 使用细胞特异性报告小鼠分析RGC与各种常驻视网膜细胞的相互作用。