Next, we examined whether iMGL maturation can be achieved with direct contact with the CNS environment. Therefore, iMGLs were cultured with rat-hippocampal neurons (21 DIV) to assess how iMGLs respond to neuronal surface cues (Figure 5A). Rat-hippocampal neurons were used because they readily form synapses in culture and can be generated with limited variability. iMGLs were subsequently separated from neurons by FACS with human specific CD45 and CD11b antibodies and profiled at the transcriptome level (Figure 5B). Differential gene expression analysis revealed that neuronal co-culturing upregulated 156 and downregulated 244 iMGL genes (Figure 5C,D). FFAR2 and COL26A1 are two genes differentially expressed in iMGLs cultured with only defined factors and indicate a developmentally primed microglia profile. In contrast, co-culturing microglia with neurons increased expression of Siglec11 and 12, human-specific sialic-acid binding proteins that interact with the neuronal glycocalyx, function in neuroprotection, and suppress pro-inflammatory signaling, and thus maintain a microglia homeostatic state (Linnartz-Gerlach et al., 2014; Wang and Neumann, 2010). Additionally, we saw increased expression of microglial genes CABLES1, TRIM14, MITF, MMP2, and SLC2A5. Overall, these results implicate both soluble and surface CNS cues as factors in microglia maturation (Biber et al., 2007)(Figure 5E,F).
