By day 38, iMGLs resemble human microglia, but not monocytes nor macrophages by cytospin/Giemsa staining (Figure 1G) and express many other microglial-enriched proteins including Mertk, Itgb5, Cx3cr1, Tgfbr1, and Pros1 protein (Figures 1H and 4S). Like murine microglia development in vivo, iMGLs developing in vitro express PU.1, TREM2, and CD11bint/CD45low (Figure 1Aiii, D–F). As iMGLs mature in vitro, they also become more ramified, similar to microglia in vivo (Figure 1Aiv, 1H). Furthermore, purinergic receptor P2ry12 and Trem2 co-expression was enriched in iMGLs when compared to monocytes and quantification reveals our protocol yielded iMGLs of high purity (>97.2%, n=5) (Figures 1I and S2A, B). Genomic integrity was also maintained over the course of differentiation. Assessing copy number variants across all chromosomes demonstrated that extra chromosomal fragments were not acquired by iMGLs when compared to their respective iPSCs (n=6, Figure S1D, E). A comparison of a representative differentiation across the entire probeset (n=383, nCounter® Human Karyotype Panel) revealed a high correlation between the iPSC and iMGL genomes (r2 > 0.92, Figure S1F). Importantly, this protocol typically yielded 30–40 million iMGLs from one million iPSCs, suggesting that our approach can be readily scaled-up for high content screening.
