Abstract
Purpose :
Loss of retinal ganglion cells (RGCs) is the final common end point in glaucoma and other optic neuropathies, ultimately leading to irreversible vision loss in millions of people worldwide. Efforts to replace RGCs with pluripotent stem cell-derived therapy have faced hurdles due to the inadequate integration and proper pathway projection of transplanted RGCs. Endogenous RGC regeneration from Müller cells presents a promising alternative, but mammalian retinas lack regenerative capacity. Hence endogenous RGC regeneration in mammals has long stood as a significant challenge. This study introduces a chemical-based method to stimulate endogenous RGC genesis and subsequent vision rescue in the injured retina.
Methods :
A combination of six chemicals along with two additional factors was injected into the eyes of mice previously injected with NMDA, allowing observation of subsequent Müller cell responses including proliferation, migration, and differentiation through immunofluorescence. To track cell lineages, an AAV2-GLAST-GFP reporter and GLAST-CreERT;Ai9 mice were utilized following the injection of the chemical cocktail. Vision function recovery was assessed using pattern electroretinogram (ERG), pattern visual evoked potential (VEP), Visual Cliff tests, and light aversion behavior tests in a model of NMDA-induced retinal ganglion cell (RGC) injury spanning from seven days to over two months. Furthermore, human primary Müller glia and fibroblasts were subjected to the chemical cocktail for in vitro reprogramming, with analysis conducted through immunofluorescence, quantitative PCR (qPCR), single-cell RNA sequencing (seq), and single-cell ATAC-seq.
Results :
we report a small molecule cocktail that causes endogenous Müller proliferation, migration, and specification to newly generated chemically induced RGCs (CiRGCs) in NMDA injured mice retina. Notably, regenerated CiRGCs extend axons towards optic nerve, and rescue vision post- NMDA treatment. Moreover, we successfully reprogrammed human primary Müller glia and fibroblasts into CiRGCs using this chemical-only approach, as evidenced by RGC-specific gene expression and chromatin signature. Additionally, we show that interaction between SOX4 and NF-kB determine CiRGC fate from Müller cells.
Conclusions :
we unveil a novel chemically mediated paradigm for endogenous RGC regeneration from Müller cells that can rescue vision in a RGC injury mice model of optic nerve diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.