Fig. 6

Reciprocal biological effects in co-culture of hiMGLs and primary neurons. A Schematic diagram of the co-culture system. B Representative images of immunostaining with IBA-1 and βIII-tubulin in co-culture or hiMGLs alone. After co-culture with primary neurons for 1 week, hiMGLs exhibited a more ramified morphology, which was not observed in hiMGL culture alone. In addition, hiMGLs in co-culture showed a larger area than those in single culture (n = 32 in single culture, 26 in co-culture; 3 independent experiments). Scale bar = 15μm. ***p < 0.001. C Mouse primary neurons were transfected with actin-GFP plasmid and then immunostained with anti-STEM121, IBA1, and GFP antibodies. Scale bar = 50μm. D hiMGLs showed attaching to axons and dendrites. Scale bar = 10μm. E The number of dendritic spines was increased in co-culture with hiMGLs. High-magnification images showed that neurons co-cultured with hiMGLs had more matured spines (mushroom and so on), while neurons in single culture had much less mature dendritic spines. Scale bar = 2μm. Spines were categorized into six groups and quantified. Quantification showed much more spines in the co-cultures than single cultures (n = 25 in single culture, 35 in co-culture; 3 independent experiments). ns, not significant; *p < 0.05. All data are expressed as mean ± SEM. F Primary neurons co-cultured with hiMGLs showed large spikes and reduced frequency. Representative images of calcium response in the co-culture (hiMGLs) or neurons alone (CTRL). Quantification of maximum ΔF/F and spike frequency indicated that hiMGLs could raise the regularity of neuron’s activity (n = 16 in single culture, 18 in co-culture; 3 independent experiments). *p < 0.05; **p < 0.01. All data are expressed as mean ± SEM. SEM, standard error of the mean