Figure 4. hPSC-derived microglia cultured with hPSC-derived astrocytes and neurons builds a functional human tri-culture system that allows for the modeling of the neuroinflammatory axis in vivo.

A) Schematic of tri-culture differentiation. hPSC-derived microglia, astrocytes, and neurons are initially plated at a ratio of 2:1:8 which then stabilizes to a ratio of ~1:11:20 microglia (~2–3%), astrocytes (~35%), and neurons at the time of assay (1 week). B) IF shows that hPSC-derived astrocytes are GFAP+ and some are also AQP4+. Scale bar = 100uM. C) IF shows that postmitotic hPSC-derived neurons express FOXG1. Scale bar = 100uM. D) IF shows that day 50 hPSC-derived neurons express TBR1 and CTIP2. Scale bar = 40uM. E) IF of tri-culture shows IBA1+ microglia and GFAP+ astrocytes interacting with MAP2+ neurons. F) IF of tri-culture shows minimal CC3 staining. Scale bar = 100 μM in (E) – (F) G) ELISA shows that tri-cultures (TRI) have increased C3 as compared to microglia/neuron only (M/N) cultures, which is exacerbated upon LPS treatment. C3 is not present in astrocyte/neuron (A/N) and neuron only (N) cultures. n = 4 samples (distinct culture supernatants), 1-way ANOVA with Tukey’s post-hoc test, **** p < 0.0001, F = 53.10, df=5; ** p= 0.0039, F=16.41, df = 5. Error bars = SD, center = mean. H) ELISA shows that C3KOA tri-cultures have less C3 as compared to WT tri-cultures but more than M/N cultures. C3KOM cultures have minimal C3. Upon LPS treatment, C3KOA tri-cultures have less C3 release as compared to WT tri-cultures; C3KOM again have minimal C3 release. n=4 samples (distinct culture supernatants), 1-way ANOVA with Tukey’s post-hoc test, **** p< 0.0001, ** p=0.0032, F= 53.10, df=5 for baseline tests; ** p= 0.0028, F=16.41, df = 5. Translucent bars (TRI, M/N) represent data originally presented in (G). Error bars = SD, center = mean. I) C3 (1ug/mL) induces IL-6 and TNF-a in wildtype microglia but not C3KO microglia. GM-CSF, IFN-g, and IL-1B are in contrast not induced upon C3 addition. ** p= 0.0022; *** p=0.0005, n= 3 cell culture supernatants, 1-way ANOVA with Sidak’s post-hoc test, Error bars = SD, center = mean. J) TNF (100ng/mL) but not IL-6 (100ng/mL) added for 48 hr to astrocytes induces C3 expression normalized to untreated astrocytes. n=2 independent experiments. K) Neuroinflammatory loop in tri-culture is initiated by LPS-activated microglia signaling to astrocytes which signal back to microglia leading to increased C3 release. Mediators include TNF secreted by stimulated microglia inducing C3 in astrocytes, and C3 secreted by both astrocytes and microglia inducing C3 in microglia.