By Alexandra Brodin
Scheie Vision Annual Report
Venkata R. M. Chavali, PhD, Assistant Professor of Ophthalmology at the University of Pennsylvania (UPenn), recently led a study to develop a promising new methodology for differentiating stem cells into retinal ganglion cells (RGCs) in vitro. This methodology has the potential to lead to more targeted treatments for glaucoma, the leading cause of irreversible blindness worldwide.
A New Vision for Treating Glaucoma
Primary open-angle glaucoma (POAG), the most common form of the disease, is characterized by chronic, progressive degeneration of the optic nerve, a cord-like structure responsible for carrying neural signals from the eyes to the brain. Damage to the optic nerve is caused by the death of retinal ganglion cells (RGCs), which are neurons located in the retina. The axons of RGCs collectively form the optic nerve.
Current therapeutic strategies for glaucoma focus on management of intraocular pressure (IOP) to slow disease progression. However, even if diagnosed in early stages, approximately 30% of patients continue to worsen despite IOP-lowering treatments. This suggests that more research is needed on additional underlying disease mechanisms independent of elevated IOP, which, once understood, could provide further therapeutic targets for glaucoma.
Because human RGCs do not regenerate naturally after they deteriorate, the idea to restore or replace damaged RGCs is very attractive as a potential therapy. Stem cells provide investigators with the opportunity to generate new, viable RGCs. These RGCs can then be used to study the various factors that cause these cells to deteriorate in vitro and in mouse models.
In recent years, vision scientists have found that RGCs can be generated using induced pluripotent stem cells (iPSCs), which are somatic cells that have been reprogrammed to an embryonic-like state. Researchers can guide iPSCs toward any type of cell lineage that may be required for experimental and therapeutic purposes, including the RGC lineage. The differentiation of iPSCs toward the RGC lineage allows researchers to study how to slow RGC death in glaucoma patients and reverse degeneration of the optic nerve by restoring or replacing damaged RGCs.
Generating Retinal Ganglion Cells
Over the past decade, different methods for generating RGCs from embryonic stem cells and iPSCs have emerged. One example is the use of a three-dimensional (3D) culture such a retinal organoid for the generation and isolation of RGCs. However, developing the retinal organoid generally requires a significant amount of time (approximately 40 to 60 days). This method also generates extraneous material, from which the RGCs must be selectively removed, leading to manual bias, lower reproducibility, and ultimately a diminished yield.
Dr. Chavali’s study, published in Nature Scientific Reports in 2020, builds on prior attempts to generate RGCs by creating an efficient and reproducible methodology that avoids the problems found in other protocols. “I wanted to generate a protocol that was efficient, highly reproducible, had limited manual selection and yields highly pure RGC cultures,” said Dr. Chavali. To conduct this research, he partnered with Dr. Jason Mills, who was then Director of the Induced Pluripotent Stem Cell (iPSC) Core at the Center for Advanced Retinal and Ocular Therapeutics (CAROT).
Dr. Chavali began working on stem cell-based methods of generating RGCs four years ago, inspired by the National Eye Institute’s Audacious Goals Initiative. This program encouraged researchers to propose innovative cross-disciplinary projects to restore vision in the retina.
The methodology that Dr. Chavali’s team developed uses two-dimensional (2D) cell cultures and does not require manual separation of cell clusters. The process involves two main stages. In the first stage, iPSCs are differentiated into retinal progenitor cells (RPCs), an intermediary cell type along the RGC lineage. “The uniqueness of our protocol is that we generate almost 100% of the RPCs from iPSCs,” said Dr. Chavali. “This is a unique advantage we have going into the second step.”
In the next stage, they used small molecules and proteins to inhibit several pathways, including BMP, TFG- (SMAD), and Wnt, to mature the RPCs towards the RGC lineage. To avoid the need for manual selection of cells through this process, Dr. Chavali’s team used a cross-hatching technique, which aids in the formation of RGCs with less variability and is distinct from other studies of this kind. The entire two-stage process takes only 35 days to complete and has been shown to yield a highly pure and robust population of RGCs.
The relatively short time frame of RGC differentiation and very high yields (up to 95% pure) are two significant advantages of this methodology. Another advantage is the absence of manual RGC separation from a retinal organoid, which can prevent damage to the newly generated RGCs. Currently, Dr. Chavali is investigating neuroprotective compounds (which work to protect cells from damage) to further help RGCs survive for long periods of time and tolerate stress more effectively.
This novel protocol is an important step towards the development of more targeted and effective treatments for glaucoma. Moving forward, Dr. Chavali plans to continue working to further improve the iPSC-RGC maturity by culturing them along with retinal astrocytes and other retinal cell types. He also intends to use this methodology to study genetic mutations that lead to RGC loss in patients and work towards a targeted therapy for patients whose glaucoma has a genetic basis.
In order to translate these findings into a strategy for treating glaucoma, Dr. Chavali’s lab is studying the molecular mechanisms of RGC death by applying oxidative stress and other conditions to simulate RGC loss in vitro. In collaboration with Katherine Uyhazi, MD, PhD, who was recently appointed as an Assistant Professor of Ophthalmology at UPenn, he is testing potential treatment strategies in mouse models of glaucoma by injecting the purified iPSC-RGCs. These experiments are currently ongoing with very encouraging preliminary results.
“Finding out ways to help patients with glaucoma is very important to me,” said Dr. Chavali. “I’m confident that our methodology of generating RGCs is a vital tool we can use to investigate mutations that cause RGC loss and offer a targeted therapy to patients with glaucoma.”
Image of retinal ganglion cells.