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.

项目摘要 功能性视网膜神经节细胞（RGC）的替换可以使视力恢复到数千万 因青光眼和其他视神经病变而失明的人。建立技术 用于RGC与多能干细胞的分化和长距离的实现 视神经内的内源性轴突再生支持RGC更换的承诺 疗法。但是，临床翻译需要显着改善功能 将移植的RGC的整合到宿主的视网膜神经通路中。为了研究 通过移植神经元的突触发生，我们提出了开发和验证创新的， 多功能，敏感，实验工具，利用小麦胚芽的透射性转运 凝集素（WGA）蛋白融合到CRE重组酶以使CRE依赖性表达 突触整合供体神经元中的记者。标签耐用，促进下游 诸如单细胞分离和转录组分析等应用以及双向的应用 在突触前或突触后 - 宿主神经元伴侣的标签。在这里，我们建议 建立与高效重组一起使用的人类多能细胞系 AAV向量报告移植RGC的功能性视网膜。由于表达 荧光报告基因组重组是自动的，该工具将有助于实时 检测体内功能性神经元整合。我们提出了一个短波长（蓝色） 突触发生的荧光记者，这将与额外的红色和绿色兼容 多色显微镜和眼镜检查的荧光。我们将表征 使用组合表达并验证此突触发生报道工具的特异性 高分辨率共聚焦显微镜，突触机械的免疫标记和单细胞 视网膜扁木内电生理学。此外，我们将研究 使用定制的多色自适应光学扫描激光器在体内移植的人RGC 眼镜镜。该仪器在XY平面内提供了亚细胞分辨率， 通过Z堆积与视网膜的深度歧视。生命眼睛的纵向成像 - 因此，移植将使供体RGC树突的相关性靶向 内部丛状层，并报告功能突触发生。我们将表征 结构事件导致供体RGC的视网膜整合。这项工作将提供 未来实验的基础，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 使用细胞特异性记者小鼠，与各种居民视网膜细胞进行RGC相互作用的分析。