Differentiation of iMGLs from patient-derived iPSCs. (A) Schematic of differentiation of human patient-derived BRAF^V600E/WT iPSCs into microglia. iPSCs were first differentiated into iHPCs for 12 days and then further differentiated into iMGLs for 24 days, followed by 4 to 10 days of iMGL maturation (B-E) Bar plot showing the differentiation efficiency of iHPCs toward iMGLs at day 5 of iMGL maturation of 4 independent experiments (except for panel B in which n = 11) determined by expression of lineage markers CD11b and CD45 (B), fractalkine receptor CX3CR1 (C), CD115 (macrophage colony-stimulating factor receptor) (D), and CD11c (E). (F) Bar plot showing the expression of LCH surface markers CD1a and CD207 (G) via flow cytometry of 6 or 4 independent experiments, respectively. Paired parametric Student t test was used when variance was significant between groups, ∗P < .05, ∗∗P < .01. (H) UMAP of 16 818 single living cells sequenced for scRNA-seq; 5 samples (3 × BRAF^V600E/WT and 2 × BRAF^WT/WT) were collected at day 5 of iMGL maturation. Unsupervised clustering identified 6 iMGL clusters named iMGL1-6. (I) Bar plot showing the distribution of BRAF^V600E/WT and BRAF^WT/WT cells in the different clusters. Frequencies of <2% are not shown in the bar plot. (J) A microglia marker score was calculated based on the expression of 12 genes (AIF1, C1QA, CSF1R, CD74, C3, CX3CR1, MERTK, P2RY12, TREM2, TYROBP, ITGAM, and ITGAX), commonly used as microglia marker genes. (K) Enrichr⁶⁴ analysis for specific markers per cluster of the iMGL data set and (L) for genes upregulated in BRAF^V600E/WT iMGLs in comparison to BRAF^WT/WT iMGLs. Letter in brackets in panels K-L indicates the database for each ontology/pathway of the heat map or bar plot, respectively: WikiPathway 2023 (W), HDSigDB Human 2021 (H), DisGeNET (N), GO_Biological_Process_2021 (G), MSigDB_Hallmark_2020 (M), and KEGG_2021_Human (K).