Abstract
Purpose :
RGCs are highly compartmentalized cells, and previous studies have demonstrated that the degeneration of RGCs in diseases such as glaucoma occurs in a compartmentalized manner, with responses to injury occurring through different mechanisms in axonal vs. somatodendritic compartments. Previous studies have demonstrated the derivation of RGCs from human pluripotent stem cells (hPSCs) to model neurodegerative features, yet they have failed to account for the compartmentalized properties of RGCs. Thus, the goals of this study were to establish a novel, microfluidic-based platform for the analysis of RGC compartmentalization in health and disease states.
Methods :
hPSC-derived RGCs were seeded into microfluidic chips to recruit and isolate RGC axons apart from the somatodendritic compartment. Initial studies explored axonal outgrowth as well as the compartmentalization of axons and dendrites via immunocytochemistry. Next, we explored the differential response between isogenic control and glaucoma-associated OPTN(E50K) RGCs in their respective axonal and somatodendritic compartments, followed by an analysis for changes in axonal transport. Finally, we established models to uniquely orient astrocytes along the proximal axonal compartment, including modulation of astrocyte reactivity as a pathological feature of RGC neurodegeneration.
Results :
Growth within microfluidic chips allowed for more robust growth and maturation of RGCs, including long-distance axonal projections as well as proper compartmentalization of axonal vs. somatodendritic compartments, based upon expression of SMI-312 and MAP2, respectively. RGCs with the glaucoma-associated OPTN(E50K) mutation exhibited a specific deficit in axonal outgrowth compared to isogenic control RGCs, which was also associated with a decreased rate of axonal transport. Finally, upon introduction of astrocytes onto the proximal axonal compartment, the induction of astrocyte reactivity led to the onset of neurodegenerative RGC phenotypes.
Conclusions :
These results represent the first application of hPSC-RGCs in a manner that effectively recapitulates the highly compartmentalized properties of RGCs. Taken together, these results should profoundly impact future studies, providing a much more physiologically-relevant in vitro model for RGC development and degeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.