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. 2020 Sep 17;217(12):e20200474. doi: 10.1084/jem.20200474

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© 2020 Liu et al.

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Figure 1. — Integrating AD-associated genetic variations into an hMGL model. (A) Schematic depiction of AD-associated risk variants characterized. SNP variants for CD33 and INPP5D, as well as R47H, A528T, and R744X coding variants for TREM2 and SORL1 (SORLA) are marked in red. ITIM, immunoreceptor tyrosine-based inhibitory motif. (B) Workflow pipeline, for generating and characterizing AD-associated mutations in hMGLs. AD-associated coding or noncoding SNPs are introduced into corresponding genomic loci in human H9 ESC lines by CRISPR-Cas9 editing. Each line was characterized for targeted mutations and off-targeting variation before differentiation and maturation into hMGLs. hMGLs were subjected to multi-omic (RNA-seq, ATAC-seq, ChIP-seq, and label-free proteome) analysis, and functional characterization as indicated. (C) Isogenic microglial differentiation scheme used in this study. ESCs were differentiated into HPCs for 10 d, where CD43⁺ iHPCs are sorted (FACS plots) and cultured in serum-free media with MCSF, IL-34, TGF-β, and insulin; CD43 (green), CX3CR1 (red), Iba1 (purple), and DAPI (blue) staining is shown for HPCs at 10 d in vitro (DIV). Cells were differentiated to microglia for an additional 25 d, whereby maturation was induced by the addition of CD200 and CX3CL1. hMGLs were stained for TREM2 (red), CD43 (green), Iba1 (purple), and DAPI (blue) and compared with HPCs (bottom panels), or TMEM119 in hMGLs (red, bottom right) as indicated. Scale bars represent 100 µm (H9, left panel), 50 µm (mature hMGLs, right panel), and 20 µm (all fluorescence images). (D) Heatmap depicting RNA-seq profiles from human microglia (red; Galatro et al., 2017; GSE99074, red), hMGLs from this study (purple), iMGLs (Abud et al., 2017; GSE117829, green). (E) 3D PCA of hMGLs (this study, purple), iMGLs (GSE117829, turquoise; GSE89189, dark blue), human fetal microglia (GSE89189, green), human adult microglia (GSE89189, light blue), myeloid dendritic cells (GSE89189, light yellow), monocytes (GSE89189, gold). PCA reveals that hMGLs cluster closely with iMGLs and human adult/fetal microglia, and are distinct from myeloid cells.