Neurons, astrocytes, and endothelial cells in the brain interact with microglia to influence gene expression and function. Our differentiation protocol attempted to recapitulate CNS cues present in the brain by including signals derived from these other cell types including CX3CL1, CD200, and TGFβ. Whole transcriptome RNA-seq analysis confirmed the importance of these factors for establishing microglia in vitro (Figures S5 and S6). TGFβ, a glia-derived cytokine, is needed for murine microglia development in vivo and in maintaining the microglial-specific transcriptome signature (Abutbul et al., 2012; Butovsky et al., 2014; Schilling et al., 2001). Differential gene expression analysis confirmed TGFβ’s role in maintaining the human microglia transcriptome signature; 1262 genes were differentially expressed in iMGLs with TGFβ, whereas 1517 genes were differentially expressed in iMGLs after TGFβ removal (24 hours). Many of the differentially expressed genes are identified as core microglial signature targets including P2RY12, TGFβR1, and CD33, and transcription factors EGR1 and ETV5, and APOE (Figure S5A–C). Examination of gene ontology highlight neurodegenerative disease pathways including AD, Parkinson’s, and Huntington’s diseases that are TGFβ dependent (Figure S5D). Furthermore, removal of
