Figure 5.

hPSC-derived reactive astrocytes induce retinal ganglion cell neurotoxicity and hyperexcitability

Astrocytes were co-cultured with RGCs, in either direct-contact or transwell cultures and induced to a reactive state through incubation with IL-1α, TNF-α, and C1q for 2 weeks. Representative images of RGCs (BRN3b:tdTomato) co-cultured with control or reactive astrocytes, in direct co-cultures (A and B), or transwell cultures (C and D).

(E and F) Reactive astrocytes promoted a reduction in the number of primary neurites and total neurite extension in RGCs, in both direct-contact and transwell culture systems.

(G) The overall complexity of RGC neurite outgrowth, assessed by Sholl analysis, was significantly decreased in both direct-contact and transwell culture systems. ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 in (E) and (F). ^∗∗∗∗p < 0.0001, RGCs co-cultured with control astrocytes vs. reactive astrocytes in contact-dependent cultures, and ^####p < 0.0001, RGCs co-cultured with control astrocytes vs. reactive astrocytes in transwell cultures, in (G). One-Way ANOVA followed by Šídák’s multiple comparisons test with selected pair.

(H–J) Upon whole-cell patch-clamp recording, RGCs were less likely to be spontaneously active in co-culture with reactive astrocytes compared to controls yet fired more action potentials upon delivery of a depolarizing current.

(K–M) RGCs co-cultured with reactive astrocytes had a higher frequency of action potentials fired, while they also exhibited a significantly lower action potential current threshold and capacitance. ^∗p < 0.05 and ^∗∗∗∗p < 0.0001. Scale bar: 100 μm. Data represent mean values ± SEM.