Fig. 6

Administration experiment of neutralizing antibodies of TNF-α and IL-1α with ASD-GM-CSF MΦ. A Representative images at DIV56 of immunostaining of MAP2 + dendrite co-cultured with ASD-GM-CSF MΦ and simultaneous administration of neutralizing antibodies for TNF-α, IL-1α, or both, or the corresponding isotype antibodies (IgG) at a concentration of 1 μg/ml. All neurons were induced from 201B7 hiPSC line, and co-cultured with macrophages of ASD1 were used in these images. Scale bar: 100 μm. B–F Results of (B) total length of MAP2 + dendrite, C average length of MAP2 + dendrite, D max length of MAP2 + dendrite, E MAP2 + dendrite count, and F branch point count of MAP2 + dendrite. The shortening of MAP2 + dendrites observed in co-culture with ASD-GM-CSF MΦ was prevented by the simultaneous administration of neutralizing antibodies. B Brown–Forsythe ANOVA test, F (3.00, 120.7) = 16.68, p < 0.0001, with post hoc multiple comparison test of Dunnett’s T3. C–D One-way ANOVA test with post hoc Tukey’s multiple comparison test (C) F (3, 139) = 9.166, p < 0.0001, D F (3, 139) = 11.37, p < 0.0001, E F (3, 139) = 0.2045, p = 0.8931. F) Brown–Forsythe ANOVA test, F (3.00, 127.6) = 10.12, p < 0.0001, with post hoc multiple comparison test of Dunnett’s T3. n(ASD-GM-CSF MΦ + IgG) = 33 fields of 13 independent dishes from five times differentiations of two control healthy hiPSC lines each, n(ASD-GM-CSF MΦ + anti-IL-1α Ab) = 36 fields of 16 independent dishes from five times differentiations of two control healthy hiPSC lines each, n(ASD-GM-CSF MΦ + anti-TNF-α Ab) = 36 fields of 16 independent dishes from five times differentiations of two control healthy hiPSC lines each, and n(ASD-GM-CSF MΦ + 2Abs) = 38 fields of 16 independent dishes from five times differentiations of two control healthy hiPSC lines each. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001