Modeling retinal ganglion cell axonal pathology associated with glaucoma using human pluripotent stem cell-based microfluidic platforms

Retinal ganglion cells (RGCs) are highly compartmentalized cells, and previous studies have demonstrated that RGC degeneration in glaucoma occurs in a compartmentalized manner, with distinct injury responses in axonal and somatodendritic compartments. Thus, the goal of this study was to establish a novel microfluidic-based platform for the analysis of RGC compartmentalization in health and disease states. Human pluripotent stem cell (hPSC)-derived RGCs were seeded into microfluidics, enabling the recruitment and isolation of axons apart from the somatodendritic compartment. Initial studies explored axonal outgrowth and compartmentalization of axons and dendrites. We then compared the differential response of RGCs differentiated from hPSCs carrying the OPTN(E50K) glaucoma mutation with isogenic control RGCs in their respective axonal and somatodendritic compartments, followed by analysis of axonal transport. Further, we explored the axonal transcriptome via RNA-seq, focusing on disease-related axonal differences. Finally, we established models to uniquely orient astrocytes along the axonal compartment combined with modulation of astrocyte reactivity as a pathological feature of neurodegeneration. Overall, RGC culture within microfluidic chips allowed enhanced cell growth and maturation, including long-distance axonal projections and proper compartmentalization. OPTN(E50K) RGCs exhibited axonal outgrowth deficits as well as decreased rate of axonal transport, compared to isogenic controls. Finally, upon introduction of astrocytes into the proximal axonal compartment, the induction of astrocyte reactivity led to the onset of neurodegenerative phenotypes in RGCs. These results represent the first study to effectively recapitulate the highly compartmentalized properties of hPSC-derived RGCs in healthy and disease states, providing a more physiologically relevant in vitro model for neuronal development and degeneration. RNA was extracted from the somatodendritic and axonal compartments of the microfluidic devices and RNA-sequencing was performed to assess transcriptional differences between (1) the somatodendritic and axonal compartments of RGCs, as well as (2) within the axonal compartment of OPTN(E50K) RGCs versus isogenic controls (axon-sequencing).