Cross-species single-cell transcriptomic analysis reveals factors limiting human Müller glial-derived retinal regeneration

Abstract

Purpose : Reactive Müller glial cells in humans do not display the regenerative characteristics of those in zebrafish. We performed the first single-nucleus RNA sequencing of retina from patients with primary open-angle glaucoma (POAG) and controls to characterize the human Müller glial response to injury.

Methods : Nuclei were isolated from trephene punches of the macula taken from postmortem eyes and sequenced. Data was integrated and analyzed after quality control with Seurat v3. Sequencing findings were confirmed by RNAscope and standard immunofluorescence. Human retinal explants were cultured with and without N3B1P3C, an inhibitor of Lats kinase in the Hippo/Yap pathway. Proliferation was measured using AlexaFluor nuclear localization of EdU indicating synthetic nucleotide uptake.

Results : After quality control and data integration, 17,401 nuclei were isolated from 26,471 original droplets, derived from macular samples of 4 patients without retinal disease and 3 patients with POAG. The proportion of retinal ganglion cells in glaucomatous retina was significantly lower than that in healthy retina (p=0.024). An activated subpopulation of Müller glia was identified in both healthy and glaucomatous retina by cell clustering. Cross-species analysis comparing zebrafish and humans identified YAP1 activation as a differentiator between zebrafish and human glial activation. Human retinal explants cultured with N3B1P3C demonstrated significant proliferation of GS+ Muller cells (p=0.044).

Conclusions : Our data, including the first single-nucleus RNA sequencing of glaucomatous retina in humans, identify an activated subpopulation of human MGCs with transcriptomic signature similar to regenerative MGCs in other species, but limited by molecular brakes such as Hippo/Yap pathway factors. By inhibiting these brakes with a small molecule (N3B1P3C), we induced proliferation of Müller glia in human retinal explants for the first time. Further manipulation of endogenous activation pathways may allow for recruitment of a quiescent stem cell population and vision-restoring therapies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Single-nucleus transcriptomic analysis of glaucomatous retina tissue. RGC: retinal ganglion cell; MGC: Müller glial cell; aMGC: activated Müller glial cell.

 

Human Müller glia decreased YAP1 expression upon activation as opposed to increased expression in zebrafish. FC: fold change upon Müller cell activation.