Chemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs)

Zhang, Wei
Fan, Yan
Chavala, Sai
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Chemical Reprogramming of Mouse and Human Müller glia into Retinal Ganglion-Like Cells (RGCs) Yan Fan, Wei Zhang, Sai Chavala Department of Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, UNT Health Science Center, Fort Worth, TX 76107 Purpose: Glaucoma is a leading cause of irreversible blindness with increasing prevalence as the population ages. Glaucoma is characterized by loss of retinal ganglion cells (RGCs), and current therapies, whether surgical, pharmacological, or neuroprotective, do not reverse the degeneration. Stem cell approaches to replace lost RGCs are a viable option. However, the use of stem cells for RGC replacement currently faces several barriers: 1) absence of a safe, non-immunogenic, and ethical stem cell source of RGCs, 2) inefficient differentiation protocols that can take more than 40 days, and 3) RGC donor integration into the degenerate host retina are major issues that need to be overcome to be considered for clinical use. A radically new approach to restoring vision for glaucoma patients that can overcome these limitations would be to reengineer a resident cell in the retina, such as Müller glia, that could serve as a reservoir for new RGCs, and avoid the need for cell transplantation. In this study, we take the first step in realizing our long-term goal of using a chemically reprogramming strategy to replace lost RGCs and restore vision.

Materials and Methods: Primary Müller glia were isolated from mouse or human retina. Small molecules were purchased from Sigma or Cayman. For the in vitro studies, Müller glia were fixed after small molecule conversion with 4% PFA. Immunofluorescence staining was performed to detect RGC specific markers: Brn3a, Brn3b, ISL-1, RBPMS, and Tuj1. Total RNA was isolated and subjected for real-time PCR for detection of neuronal marker NeuN and RGC markers: Brn3a, Brn3b, ISL-1, NefH, and Nefl. For the in vivo studies, intravitreal injection was performed to deliver small molecules and other growth factors. BrdU was injected intravitreally or intraperitoneally to label proliferating and regenerating cells. 24 hours after the final injection, eyes were enucleated, fixed, and embedded for frozen sections. Immunofluorescence staining was performed to detect proliferation with BrdU and Ki67. After 7, 21, and 35 days of final injection, BrdU and RGC markers were co-stained to detect newly generated RGC-like cells. Results: Both mouse and human Müller glia can be converted to RGC-like cells within 24 hours in vitro. In vivo injection of small molecule and growth factor cocktail into the vitreous stimulated cell proliferation, which was located between the outer nuclear layer and the inner nuclear layer where the cells stained positive with GFAP. After 7 days, BrdU positive cells migrated to the inner edge of INL. After 21 days, BrdU positive cells reached the ganglion cell layer (GCL) but no co-staining with a RGC marker was observed at this time point. Strikingly after 35 days, we detected co-stained BrdU and ISL-1 or RBPMS in the GCL. There was no retinal toxicity observed from the small molecule injection. Conclusion: Our small molecule cocktail is highly efficient in converting mouse and human Müller glia to RGC-like cells in vitro, enabling us to generate RGC-like cells in approximately one day with more than a 90% conversion efficiency. The small molecule cocktail can be injected intravitreally to stimulate cell proliferation and RGC regeneration in vivo. Additional retinal toxicity testing will be needed to evaluate the safety profile of the intravitreal small molecule cocktail.

Research Appreciation Day Award Winner - 2019 North Texas Eye Research Institute, Postdoctoral Poster Presentation Award - 1st Place
Research Appreciation Day Award Winner - 2019 Graduate School of Biomedical Sciences, Postdoctoral Poster Award - 1st Place