Cell Replacement in Glaucoma: Making Mature RGCs
Retinal ganglion cells (RGCs) are highly specialized neurons, which connect the photosensitive part of the eye with the appropriate targets in the brain. While RGCs exhibit limited plasticity and can form new cell-to-cell connections throughout our life, their number is fixed, and no new cells arise postnatally. Thus, any stress factors leading to ganglion cell death, such as high intraocular pressure in Glaucoma, trauma or drug toxicity, result in irreversible vision loss. In our pilot studies we have demonstrated that mouse RGCs may be produced from renewable cell source – pluripotent/adult stem cells and successfully delivered into the mouse eye. In this proposal we aim to improve the donor, stem cell-derived RGCs more mature and developmentally closer to the “real” RGCs. That should significantly increase the transplantation success, leading to potential therapy development.
My lab is committed to the development of cell replacement therapies for glaucoma. With the support from the BrightFocus National Glaucoma Research Program we are able to get one step closer to our goal by looking into the maturation of stem-cell derived donor RGCs in our transplantation studies. In this proposal we plan to build upon state-of-the art approach for the retinal organoids differentiation and utilize novel technological approaches for growth factor delivery and cell analysis. We plan to employ polyhedrin-based growth factor delivery system, that can maintain growth factor levels in vitro and in vivo. As for the cell analysis we plan to expand the application of single-cell analysis techniques to better understand the condition of the cell: single-cell RNA sequencing for molecular profiling and multielectrode array for functional analysis. These techniques would allow us to distinguish properties of individual cells and describe the heterogeneity of stem-cell derived RGCs. We hypothesize that retinal organoids derived from pluripotent stem cells are self-sufficient in their ability to recapitulate development and form bona fide retinal ganglion cells. We postulate that by rescuing RGC from cell death in vitro we may provide necessary time to acquire mature molecular and functional features. If our assumption is correct, improving RGC survival in stem cell-derived retinal organoid cultures should lead to enhanced maturation in vitro and improved transplant outcome in vivo. In this proposal we plan to use known intrinsic and extrinsic modulators of RGC survival as parallel approaches to study the effect of the survival on the last stages of RGC maturation from stem cells. The successful completion of the project would allow us to establish optimal conditions for RGC differentiation from stem cells. We aim to continue working on the project with the overall goal to develop a cell replacement therapy for late-stage Glaucoma, and this is an important step that would allow to define conditions for “Cell product manufacture”. The next step would be confirming those findings with human pluripotent stem cells, followed by transplantation studies in small and large animal models of Glaucoma.