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. 2022 Aug 11;25(9):1149–1162. doi: 10.1038/s41593-022-01131-4

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a, Strategy for stable integration of six transcription factors in AAVS1 and CLYBL loci by TALEN-mediated integration: The doxycycline-inducible reverse transcriptional activator (rtTA3G) is driven by the constitutive CAG promoter. Human MAFB, CEBPα and IRF8 are driven by the tet response element (TRE3G) in the AAVS1 locus. Human PU.1, CEBPβ and IRF5 are driven by TRE3G in the CLYBL locus. All transcription factors are separated from each other via T2A ribosome skipping sequences. b, Overview of the differentiation process for generating iTF-Microglia. Top, timeline with media, cytokines and doxycycline (Dox); bottom, representative phase-contrast images of cells on the indicated days. Scale bar, 100 μm. c, Expression of six inducible transcription factors during iTF-Microglia differentiation. Transcript abundance (transcripts per million, TPM) of MAFB, CEBPα, IRF8 cassette and the PU.1, CEBPβ, IRF5 cassette at day 0, day 9 and day 15 of differentiation. Mean ± s.d., n = 3 biological replicates, P values from two-tailed Student’s t-test. d, Representative immunofluorescence micrographs of iTF-Microglia on day 8 of differentiation stained for microglia markers GPR34 and IBA1. Nuclei were labeled by Hoechst 33342. Scale bar, 100 μm. e, Expression of iPSC and microglia marker genes in iPSCs and derived iTF-Microglia on day 9 and day 15 of differentiation. The heatmap displays normalized and gene-centered TPM counts for selected genes (rows) for three biological replicates of timepoints (columns). iTF-Microglia express microglia homeostatic markers and activation markers, while losing their expression of iPSC markers. Asterisks highlight microglia-selective markers. f, Principal component analysis (PCA) on the expression of microglia marker genes of iTF-Microglia, human adult ex vivo microglia⁶⁰, fetal and adult microglia¹³, human myeloid cells²⁰, other iPSC-microglia (iMG) / iPSC-microglia-like cells (iMGL)^13,18,61 and iPSCs (this study and ref. ⁶¹). Each dot reflects an independent biological sample. Colors represent the different cell types.