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. 2024 Mar 20;15:2497. doi: 10.1038/s41467-024-46695-w

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic representation of iMG differentiation protocol. Bone morphogenetic protein 4 (BMP-4), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) are used for embryoid body differentiation. Macrophage colony-stimulating factor (M-CSF) and interleukin (IL)−3 are included during PMP generation. IL-34, M-CSF, and transforming growth factor beta (TGF-β) are added during terminal iMG differentiation. Created with BioRender.com. b, c WT and C71G^+/⁻ iPSCs differentiated into iMGs. b Representative immunofluorescence images of P2RY12, TMEM119, and PFN1 for n = 3 independent differentiations. Scale bar: 100 µm. c Comparison of gene expression levels between iPSCs, PMPs, and iMGs of myeloid and microglia-enriched genes including GPR34 (*P = 0.0173, ** P = 0.0044), PROS1 (**P = 0.0040, ***P = 0.0002), P2RY12 (**P = 0.0030 for iPSC vs iMG, **P = 0.0033 for PMP vs iMG), MERTK (**P = 0.0016 for iPSC vs iMG, **P = 0.0049 for PMP vs iMG), and SPI-1 (*P = 0.0240) and the pluripotency marker SOX2 (***P = 0.0003) d Three-dimensional principal component analysis (PCA) of iMGs from this study (green; WT n = 7, C71G^+/⁻ n = 4, M114T^+/⁻ n = 3, M114T^+/+ n = 3, where n = an independent differentiation), primary human adult (yellow) and fetal microglia (navy blue) from Abud et al.⁴ iPSC-derived microglia-like cells (pink) and the intermediate hematopoietic progenitors cells (HPCs, dark orange) from Abud et al.⁴ iPSC-derived microglia-like cells (light orange) and the intermediate progenitors PMPs (light blue) from Brownjohn et al.⁵ and monocytes (dark blue) and iPSCs (gray) from Abud et al.⁴. Variance of each principal component (PC) is indicated in parenthesis along the axes. e WT and C71G^+/⁻ iMGs secrete elevated levels of IL-6 (**P = 0.0050), IL-10 (*P = 0.0156), CCL5 (**P = 0.0077) and TNF-α (*P = 0.0208) after 6 h or 24 h of 100 ng/mL LPS stimulation compared to untreated cells. Statistics were determined by two-way ANOVA and Šídák’s multiple comparisons test. No WT vs C71G^+/⁻ comparisons were statistically significant. P-values are listed only for WT cells for simplicity; all other statistical comparisons are defined in Data S1. Mean ± SEM for n = 3 independent differentiations are shown for all bar graphs, with each data point representing an individual differentiation. Source data are provided as a Source Data file.