# Retinal Ganglion Cell Subtype Specification in Human Retinal Organoids

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2022 · $46,752

## Abstract

Project Summary
 Retinal ganglion cells (RGCs) are the interneurons that transmit visual information from the eye to the
brain. Degeneration or death of RGCs results in a number of blinding conditions, including glaucoma. RGCs can
be classified into many subtypes, each with distinct morphologies, functions, and gene expression profiles. While
RGC subtypes have been identified in many vertebrate model organisms, RGC subtypes in the human retina
have not been well-characterized molecularly. Moreover, the mechanisms controlling the specification of RGC
subtypes remain poorly understood, especially in the human retina. In this project, I will study human retinas and
organoids to identify human RGC subtypes and determine mechanisms controlling their generation. My work will
yield mechanistic insights into human RGC subtype specification and facilitate the use of stem cell-derived
organoids in regenerative therapies for retinopathies like glaucoma.
 One of the major goals of this project is to molecularly classify the RGC subtypes of the adult human
retina. Based on published single cell RNA sequencing (scRNA-seq) data in human and macaque retinas, I
generated a testable gene expression code to uniquely identify each RGC subtype. I will use this gene
expression code to guide combinatorial expression analysis (i.e. immunohistochemistry and RNA FISH) and
identify human RGC subtypes. I have already distinguished three human RGC subtypes using my gene
expression code. I will also characterize the morphologies of these RGC subtypes. I will use combinatorial IHC
and the lipophilic dye DiD to molecularly and morphologically classify human RGC subtypes (Aim 1).
 A major challenge to studying human RGC biology is the limited access to genetically and
pharmacologically manipulatable human tissue. Human retinal organoids provide a powerful model to study
developing human tissue in controlled conditions. The Johnston lab advanced the use of human retinal organoids
to study the mechanisms controlling photoreceptor fate specification. I showed that organoids are a tractable
model system to study RGC biology by developing an RGC axon outgrowth assay, which demonstrated that
RGCs rapidly extend axons, recapitulating their in vivo capabilities. Furthermore, I identified two subtypes of
RGCs in organoids. To identify the repertoire of RGC subtypes in human organoids, I will assess expression
based on the testable gene expression code and complement this approach by conducting scRNA-seq over a
time course of organoid development (Aim 2). The transcription factors EOMES/TBR2, TBR1, and TBX5 have
been implicated in RGC subtype specification. To investigate mechanisms that specify human RGC subtypes, I
will utilize CRISPR mutagenesis to knock out and viral transfection to ectopically express these three
transcription factors and examine changes in RGC subtype populations in human retinal organoids (Aim 3).

## Key facts

- **NIH application ID:** 10389368
- **Project number:** 1F31EY033656-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Brian Guy
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $46,752
- **Award type:** 1
- **Project period:** 2022-03-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10389368

## Citation

> US National Institutes of Health, RePORTER application 10389368, Retinal Ganglion Cell Subtype Specification in Human Retinal Organoids (1F31EY033656-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10389368. Licensed CC0.

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