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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

基本信息

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
项目类别：
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

项目摘要

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