Induction of reactivity in human pluripotent stem cell-derived astrocytes and their contribution to retinal ganglion cell degeneration

Abstract

Purpose : Astrocytes closely associate with retinal ganglion cells (RGCs) in the nerve fiber layer of the retina and optic nerve, where they provide support for RGCs but can contribute to RGC neurodegeneration in a glaucomatous state. However, the mechanisms by which astrocytes promote neurotoxicity and contribute to neurodegeneration remain unclear. Importantly, human pluripotent stem cells (hPSCs) can serve as powerful tools for the in vitro analysis of human neurodegenerative diseases, including neuron-glia interactions.

Methods : Using hPSC-derived RGCs and astrocytes, we explored how reactive astrocytes contribute to RGC degeneration. Human pluripotent stem cells were differentiated into 3D retinal organoids or forebrain organoids for the subsequent isolation of retinal ganglion cells (RGCs) and astrocytes, respectively. The induction of a reactive astrocyte phenotype (“A1”) was promoted through incubation with a cocktail of recombinant proteins including C1q, TNFα and IL1α.

Results : Reactive astrocytes displayed profound morphological alterations exhibiting a hypertrophic profile and increased expression of reactive astrocyte-specific markers such as complement C3. Transcriptional analyses of reactive astrocytes revealed an upregulation of genes associated with the inflammatory pathway as well as cytokine signaling. Functionally, reactive astrocytes affected blood-brain barrier properties by reducing transendothelial electrical resistance and increasing barrier permeability. Moreover, the secretion of several pro-inflammatory cytokines was increased in reactive astrocytes. Consequently, the neurotoxic potential of reactive astrocytes was determined through co-cultures with hPSC-derived RGCs, in which reactive astrocytes promoted marked morphological alterations including neurite retraction and reduced neurite complexity.

Conclusions : The results of this study demonstrate that hPSC-derived astrocytes can be induced to acquire a reactive and dysfunctional profile with a predominant inflammatory and neurotoxic phenotype which leads to RGC neurodegeneration. Thus, the modulation of reactive astrocytes could offer a novel therapeutic strategy for glaucoma by restoring a more homeostatic state and reducting RGC neurodegenerative phenotypes.

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