Humans rely heavily on vision, primarily facilitated by cone photoreceptors in the macula, which provide high-definition spatial and chromatic resolution. Macular degeneration, a leading cause of blindness, affects about 20 percent of people over 45 in Wisconsin. Recent advances in human pluripotent stem cell (hPSC) engineering have made it possible to create 3D retinal organoids (ROs) that mimic the retina’s structure but have yet to demonstrate the phototransduction needed for vision. The goal of this project was to demonstrate that cone cells in lab-grown ROs can respond to light like those in a healthy human eye and test gene therapies to restore vision in patients with achromatopsia.
The researchers found that cells in lab-grown ROs had slower and less sensitive responses compared to those in mature primate eyes and confirmed the immaturity of the ROs through 3D mapping of cell connections. The lack of a retinal pigment epithelial (RPE) layer, essential for recycling vision pigments, resulted in limited light sensitivity, but adding artificial pigments improved the cells’ response to light. Using stem cells from achromatopsia patients, the team created ROs that mimicked the condition and they are now testing nanoinjection for delivery of gene therapy after unsuccessful attempts with viral vectors. Overall, this project has made significant strides in understanding and improving lab-grown retinal cells’ function.
The Challenge
Humans rely heavily on vision, which is primarily facilitated by cone photoreceptors in the macula of the retina, allowing for high-definition spatial and chromatic resolution. Macular degeneration, or cone death, is the leading cause of blindness in developed countries, affecting approximately 20 percent of people over 45 in Wisconsin. Macular degeneration is associated with increased risks of diabetes, arthritis and mobility issues. Currently, there are no treatments for macular degeneration, but recent advances in human pluripotent stem cell (hPSC) engineering have made it possible to create 3D retinal organoids (ROs) that mimic the human retina’s structure. However, these ROs have yet to demonstrate the essential function of phototransduction which is necessary for their use in transplantation therapies and disease modeling.
Project Goals
The goals of this project were to demonstrate that cone cells in lab-grown ROs can respond to light like those in a healthy human eye, to use these ROs to study how diseases affect vision and to test gene therapies to restore vision. Researchers planned to reach these goals by, first, measuring light-evoked electrical signals from individual cone cells in mature ROs and comparing them to those in healthy primate eyes. Next, they aimed to determine the anatomical and functional connections between cones and other retinal cells to see if light signals were effectively transmitted. Finally, the team sought to assess the function of cone cells in ROs derived from patients with achromatopsia, an inherited vision disorder caused by mutations in the CNGB3 gene, and test gene therapy to restore their function.
Results
The researchers made significant progress toward each of their goals. They found that the light responses of cone cells in lab-grown ROs were slower and less sensitive compared to those in mature primate eyes, similar to what is seen in developing fetal primates. A 3D map of the connections between cone cells and other retinal cells in the ROs confirmed the immaturity of the ROs as the connections were similar to those in developing fetal primate eyes. The lack of a specific layer called the retinal pigment epithelial (RPE) layer in the RO was identified to be an issue because this layer is needed to recycle the pigments needed for vision. As a result, only some of the cone cells in the ROs were sensitive to light. The team addressed this issue by adding artificial pigments to the ROs which made more cells respond to light, and they saw the most noticeable improvement around day 220 of development. These findings suggest that the cells’ need for a RPE layer changes overtime.
Researchers used stem cells from patients with achromatopsia to create ROs. Because these ROs harbor the causative mutations in the CNGB3 gene, they mimicked the condition, showing a loss of function in the cone cells without changing their overall structure.The team continues to work on optimizing their gene restoration strategies after their attempts to use a viral vector to deliver a healthy version of the gene was unsuccessful. They are currently testing a different method, called nanoinjection, to deliver the gene directly into the cells. Overall, this work has made important strides in understanding and improving the function of lab-grown retinal cells.
