# Visualizing functional retinal integration of transplanted retinal ganglion cells

> **NIH NIH R21** · JOHNS HOPKINS UNIVERSITY · 2022 · $245,625

## Abstract

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.

## Key facts

- **NIH application ID:** 10510837
- **Project number:** 1R21EY034332-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Thomas Vincent Johnson
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $245,625
- **Award type:** 1
- **Project period:** 2022-09-30 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10510837

## Citation

> US National Institutes of Health, RePORTER application 10510837, Visualizing functional retinal integration of transplanted retinal ganglion cells (1R21EY034332-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10510837. Licensed CC0.

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