Growing Human Retinas from Stem Cells to Study the Dying Neurons in Glaucoma

Robert Johnston, PhD
Johns Hopkins University (Baltimore, MD)
Year Awarded:
2019
Grant Duration:
July 1, 2019 to June 30, 2021
Disease:
Glaucoma
Award Amount:
$200,000
Grant Reference ID:
G2019300
Award Type:
Standard
Award Region:
US Northeastern
Robert Johnston, PhD

Growing Human Retinal Organoids to Study Retinal Ganglion Cell Birth and Death in Glaucoma

Summary

During glaucoma, the neurons that connect the eye to the brain die, leading to vision loss. We have learned a great deal about these cells from studies in other animals like mice and fish, yet studies directly in developing human tissue have been limited. Here, we propose to grow human retinas in a dish from stem cells to (1) determine what genes are on or off in these critical neurons, (2) develop treatments to increase the number of these neurons, and (3) study how these neurons die and develop ways to prevent their death. Our work will be the first to study these mechanisms in developing human tissue, providing insights critical for understanding glaucoma progression and therapeutic applications.

Details

My research program is dedicated to understanding how the cell types of the human retina are generated and incorporating this knowledge into the development of retinal organoid technology for visual restoration therapies.

My lab grows human stem cells into retinas in a dish. These mini-retinas are called "retinal organoids". Human retinal organoids hold huge promise for therapeutic treatments for sufferers of glaucoma. In particular, we are trying to understand the biology of retinal ganglion cells (RGCs), the cells that die during glaucoma.

The first step to understanding the utility of retinal organoids is to determine how they recapitulate normal retinal development. To achieve this goal, we are characterizing the types of RGCs generated in organoids by analyzing which genes are on or off across individual cells. The next goal is to promote the generation of RGCs. During our previous studies of human photoreceptor cells, we identified conditions that promote specialized regions of the retina that display high numbers of RGCs. We will test these conditions in organoids and look for increases in the generation of RGCs. Finally, the death of RGCs in glaucoma is a true challenge to overcome. Interestingly, RGCs die in organoids and we hypothesize that they die in a similar manner during glaucoma. We will directly compare death of RGCs in glaucoma and in organoids and tests ways to prevent death in organoids.

My lab advanced human retinal organoids as a model to study photoreceptor specification during human development. My lab is among the leaders in the world using this innovative technology to study human biology. We are in a unique position to study RGC biology with potential for glaucoma prevention and vision restoration therapy.

Our studies will directly impact our understanding of RGC biology and glaucoma. Our work will be the first to study the mechanisms of RGC specification and death in developing human tissue, providing insights critical for understanding glaucoma progression and therapeutic applications.

About the Researcher

I was born and raised in Philadelphia where I attended Drexel University, double majoring in Biology and Teaching. After graduation, I worked in the lab of Nobel Laureate James Rothman at Memorial Sloan Kettering Cancer Center, studying how parts of cells fuse. In graduate school, I found my true scientific interest, understanding how different neurons are made during development. As a graduate student in the laboratory of Oliver Hobert at Columbia University/HHMI, I identified the first microRNA (a type of gene regulator) to play a role in nervous system development. I then conducted post-doctoral research in the lab of Claude Desplan at New York Univeristy, where I studied how photoreceptors are made in the fruit fly eye. Starting in my lab at Johns Hopkins, I continued my studies of fruit fly eye development, but also extended our studies to humans. In Johnston lab, we study how photoreceptors and retinal ganglion cells are generated, using human retinal organoids, grown from stem cells, as a model. Our lab showed that this system could be used to study human biology (Eldred, et al. 2018), and are now using the system to understand neuronal development with the ultimate goal of developing therapies for glaucoma and macular degeneration.

Personal Story

My lab at Johns Hopkins University is interested in how the cells in our eye are made. We initially focused on the cells that see color. There are three types of color-detecting cells that sense red, green, or blue light. We cannot study how these cells are made in developing human babies, so we take human stem cells and direct them to become retinal tissue. These retinas grown from stem cells are called retinal organoids. One thing that amazed us was that these retinal organoids grow in a manner that is very similar to real human retinas: the cells are made at the same times, the cells look the same, and the genes work in the same ways.

Since human development takes 9 months, this posed a particular challenge - could we study human development in organoids grown in a dish on fetal timescales? We took this as a challenge and grew these organoids for a full year, checking their development at different times. We found that the blue cells were made first and the red and green cells were made later - it is as if there is a timer for when these cells are generated. What is particularly remarkable is that the growing organoid knows what to do. After initially directing the stem cells to become retinal tissue, we do not add any special chemicals or signals, the organoids make these cells themselves!

We next sought to figure out how the timer worked - how are the blue cells made first, and the red and green cells made later? Pioneering work in mice, fish, and chickens, led us to test a set of genes involved in the function of thyroid hormone. We formed the hypothesis that thyroid hormone function was low early to make the blue cells, and high later to make the red and green cells. Perhaps the most beautiful results of our work came when we manipulated thyroid hormone function. We used CRISPR to knock out the receptor for thyroid hormone and generated human organoids with only blue cells. We then added thyroid hormone and generated human organoids with only red and green cells. These striking results told us that thyroid hormone was important for making the color-sensing cells in our eyes.

There was one last challenge: if thyroid hormone controls the development of our color-sensing cells, how is it made - there is no thyroid gland in the dish!! The eye itself must control thyroid hormone levels. To answer this question, we looked at what genes were on or off during eye development for a full year. We found that genes that degrade thyroid hormone were on early to make the blue cells, and genes that activate thyroid hormone were on late to make the red and green cells.

This work is important because it is the first to use human organoid technology to study the mechanisms of human development on fetal timescales. The timescale of 6-12 months is particularly challenging since these experiments are done in antibiotic-free conditions and we must be extremely careful to prevent contamination that can destroy the organoids. It takes a special type of scientist to conduct these experiments. Kiara Eldred, Sarah Hadyniak, and Kasia Hussey, had to be very careful and mentally tough, knowing that any misstep could jeopardize their work. These scientists are very intelligent and they made smart decisions to ensure their success. Johns Hopkins is an extremely collaborative environment and this work could not have happened without help from Don Zack and Karl Wahlin.

To conclude, this work has important implications for our understanding of human development and potential treatments for vision disorders. Vision disorders had been linked to alterations in levels of thyroid hormone in premature babies previously. Our work directly shows the mechanism and cause, and suggests possible treatments to prevent these problems. The next big challenge is taking our findings and using them to provide therapy to people with visual disorders and diseases. Importantly for this project at BrightFocus, our first work set the stage for our new experiments addressing RGC biology and glaucoma. These retinal organoids are amazing tools with great promise for understanding human biology and developing therapies.

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