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. 2022 Jan 20;13:430. doi: 10.1038/s41467-022-28043-y

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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. 3 — a A construct to target the AAVS1 locus to generate rtTA-PU.1-IRES-eGFP⁺ hESC line (BC61). b Schematic showing the protocol for generating mhCOs by using hESCs (BC61). c Time-lapse imaging indicating GFP⁺ microglia over 100 min within mhCOs. Representative images with white arrowheads showing motile microglia. Time is shown in minutes. d FACS analysis of dissociated mhCOs at day 75 shows that GFP⁺ and GFP⁻ cells are sorted for performing scRNA-seq. e Cell trajectory analysis of the single-cell transcriptome of GFP⁺ and GFP⁻ cells. Cells are colored by trajectory branches and library sources. f Ratio of GFP⁺ and GFP⁻ cells in MG clusters. g Comparison of GFP expression between MG and other branches (two-sided T-test p < 2.2e-16). h Relationship between GFP/PU.1 expression and MG lineage commitment. (Top) GFP and PU.1 expression are plotted in the cell trajectory map. (Bottom) Correlation between GFP/PU.1 expression and efficiency of MG lineage commitment. Cells are sorted by GFP/PU.1 expression and divided into 100-cells bins. The number of cells in the MG branch is shown in each bin. i Top, schematic showing Aβ42-oligo-(HiLyte)-555 treatment on mhCOs. Bottom, a representative image is showing GFP⁺ microglia phagocytosing Aβ (red) in around 32 min within mhCOs. The scale bar represents 20 μm in (c) and 10 μm in (i). Imaging was repeated in organoids from three independent differentiation experiments with similar results in c and i.