A multiplexed, single-cell sequencing screen identifies compounds that increase neurogenic reprogramming of mammalian Muller glia.

Neurodegeneration in the retina of mammals causes permanent loss of vision, due to their lack of regeneration. Some non-mammalian vertebrates show robust regeneration, via the Muller glia (MG), and we have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. We designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single nucleus RNA-seq conditions into a single experiment, to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease. Harvested and then in vitro reprogrammed Muller glia were subjected to sci-Plex after various treatments with Ascl1 OE and the addition of small molecules. MG were also reprogrammed in vivo by ANT treatment and subjected to 10x scRNA-seq 21 days after injury.