By Brittany Wade 

January 18, 2023 | A new study published in the Proceedings of the National Academy of Science demonstrates the potential for lab-grown retinal neurons to reestablish synaptic function and cure neurodegenerative eye disease, according to a University of Wisconsin–Madison (UW-Madison) research team. 

The published findings are notable (DOI: 10.1073/pnas.2213418120), as no other study has indicated that retinal organoid cells—a three-dimensional organized cell cluster mimicking the retina—can regain synaptic function after being disassembled and then attached to a two-dimensional culture system. 

The cells’ ability to reconstitute function post-detachment suggests that lab-grown neural implants could potentially replace degenerate neurons and restore vision to patients experiencing blindness. 

“We wanted to use the cells from those organoids as replacement parts for the same types of cells that have been lost in the course of retinal diseases,” said David Gamm, MD, Ph.D., UW–Madison ophthalmology and visual sciences professor and director of the McPherson Eye Research Institute, in a press release. “But after being grown in a laboratory dish for months as compact clusters, the question remained—will the cells behave appropriately after we tease them apart? Because that is key to introducing them into a patient’s eye.” 

The study focused on three organoid-derived retinal cell types: photoreceptors (rods and cones), retinal interneurons, and retinal ganglion cells (RGCs). These cells belong to a neural network in the retina that processes visual information by converting light into nerve impulses interpreted by the brain. 

Abnormal changes in photoreceptors and RGCs are often an early sign of retinal degeneration, including diseases like retinitis pigmentosa—a genetic disorder that breaks down photoreceptors over time—and glaucoma, a condition associated with vision loss due to optic nerve damage (Frontiers in Cellular Neuroscience, DOI: 10.3389/fncel.2015.00395).  

Unlike non-mammalian vertebrates, humans and other mammals cannot regenerate lost neurons. Therefore, any attempts to restore sight—at least in part—would require an implant of exogenous cells or a morphological and functional shift of existing and surrounding cell types. 

Whether from exogenous or endogenous origins, all retinal neurons must form synaptic connections with other neurons to facilitate sight. A synapse, also called a neuronal junction, is the space at the end of a neuronal process or cord—typically an axon—where nerve impulses are released to affect change or action in the body. The UW–Madison team refers to synaptic activity between two neurons as a “handshake.” 

Since developing retinal organoids over a decade ago, the team has proven that organoid-derived photoreceptors behave similarly to their in vivo counterparts and can even form axons (Cell Stem Cell, DOI: 10.1016/j.stem.2022.01.002 and Cell Reports, DOI: 10.1016/j.celrep.2022.110827). “The last piece of the puzzle was to see if these cords had the ability to plug into, or shake hands with, other retinal cell types in order to communicate,” said Gamm. 

Completing the Puzzle 

The team separated individual human pluripotent stem cells (hPSCs) from 80-day-old retinal organoids (ROs) using papain, an enzyme known to nullify synaptic connections between neurons and ensure the cells begin as individual entities. A waiting period of 80 days allowed the hPSCs to differentiate into the cells of interest. 

The team used a monosynaptic tracing assay to track each neuron’s activity. Ten days after separation, approximately 5% of the hPSCs received a lentivirus containing a green fluorescent nucleic tag and other cellular machinery, including a viral binding receptor. 

Five days later, the neurons were inoculated with a replication-deficient monosynaptic retrograde rabies virus containing a red fluorescent tag. The virus was genetically engineered to penetrate mammalian cells and bind to the same viral receptor as those added to the hPSCs five days prior. Because of the fluorescent tags, these select “starter” cells would possess a green nucleus and red cytoplasm. 

As a retrograde virus, the microorganism can travel from neuron to neuron via synaptic transmission. Any new or “traced” cell with an unmarked nucleus and red cytoplasm would result from a starter cell transmitting the virus via a synaptic connection. The term “retrograde” denotes that the virus travels across the synapse in the opposite direction as traditional neurotransmitters. 

Using confocal and high-content widefield imaging, the team observed that the traced cells grew to 6.2% of the cellular population, proving that de novo retinal neurons could successfully undergo synaptogenesis and maintain full function after detachment from an organoid. 

Looking ahead, the team plans to examine if the cells can develop a more mature synaptic architecture after detachment for more extended periods. “We’ve been quilting this story together in the lab, one piece at a time, to build confidence that we’re headed in the right direction,” said Gamm. “It’s all leading, ultimately, to human clinical trials, which are the clear next step.” 

The team’s work opens the door for future human clinical trials where cultured cells can be extensively evaluated for future cell replacement therapies. Historically, preclinical studies involved transplanting human retinal neurons into limiting animal models. Now, neurons from hPSC-derived ROs can be reproduced without restrictions and perform almost identically to human in vivo cells. 

Even now, the team’s findings are making a marked difference. Gamm co-founded Opsis Therapeutics, a biotech research company adapting some of the team’s discoveries to develop replacement therapies for age-related macular degeneration.