Abstract
Purpose :
The aim of this study is to identify small molecule compounds that can reprogram retinal Müller cells (MCs) into photoreceptor cells and to evaluate the recovery of retinal function in animal models of retinal degeneration.
Methods :
In vitro, MCs were stimulated with all combinations of candidate compounds, and rhodopsin (Rho) expression was confirmed on day 7 by Polymerase Chain Reaction and immunostaining. In vivo, we used 6-week-old N-methyl-N-nitrosourea [MNU]-treated and 4-week-old rd10 mice, representative models of retinal degeneration. The optimal combination of compounds determined by in vitro screening was injected into the vitreous and the changes in Rho expression were evaluated on day 7. The origin of Rho-positive cells was also validated in td-Tomato reporter mice. PAAV.GFAP.Cre.WPRE.hGH, which is an adeno-associated virus (AAV) containing human GFAP promoter, was injected into the vitreous of 2-week-old B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice so that MCs could be visualized. On day 7 after MNU administration, co-expression with td-Tomato and Rho in the outer retina was confirmed by immunohistochemistry. Finally, the recovery of retinal function was assessed by electroretinography.
Results :
In vitro, mRNA expression of Rho in MCs increased 30-fold, and 25% of MCs expressed Rho protein 7 days after stimulation with the combination of 4 compounds: tumor growth factor-β inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and γ-secretase inhibitor. In vivo, Rho mRNA expression and the number of Rho-positive cells in the outer retina were significantly increased on day 7 after the intravitreal injection of these 4 compounds in both MNU-treated and rd10 mice. Lineage tracing in td-Tomato mice treated with MNU showed that the regenerated Rho-positive cells were originated from endogenous MCs, accompanied with recovery of Rho-derived scotopic function, which suggested Rho-positive cells may have the properties of photoreceptor cells.
Conclusions :
These results suggest that photoreceptor cells can be regenerated from endogenous retinal MCs via direct reprogramming by small molecule compounds.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.