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Efficient derivation of microglia-like cells from human pluripotent stem cells

Abstract

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell–derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

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Figure 1: Induction of primitive myelogenesis from human pluripotent stem cells.
Figure 2: Characterization of phagocytes delaminating from cystic YS-EBs.
Figure 3: pMGLs adopt ramified morphologies over time and express specific markers of microglia.
Figure 4: pMGLs cytokine profiles in response to endotoxin challenge.
Figure 5: pMGLs recapitulate the consensus signature distinguishing primary microglia from other macrophages.
Figure 6: Neural co-cultures enhance the microglial signature of pMGLs.

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Acknowledgements

The authors thank L. Freland-Gobaille, D. Fu, R. Alagappan, T. Lungjangwa and S. Elmsaouri for technical support, and all members of the Jaenisch lab for helpful discussions. We thank P. Thiru and G. Bell at WIBR's Bioinformatics and Research Computing group for help and advice with sequencing-data analysis. Confocal microscopy and wound assays were performed at the Keck Facility, with the precious help of W. Salmon. We thank P. Wisniewski and C. Zollo for help with cell sorting. We thank T. Volkert, J. Love and S. Gupta at the WIGTC for help with library preparation and sequencing. J.M. received funding from the European Leukodystrophy Association and a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation. Y.L. received funding from a Simons postdoctoral fellowship, an International Rett Syndrome Foundation (IRSF) postdoctoral fellowship and a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation. S.C. was the recipient of a Simons undergraduate scholarship and an Amgen Scholarship. G.B. was supported by H.H.M.I. Work for this project was supported by a grant from the Simons Foundation (SFARI 204106 R.J.), NIH grants HD 045022, R37-CA084198 and NS088538, the ELA foundation, the Emerald foundation and Biogen (to R.J.) and NIH 1RF1 AG042978 to L.-H.T.

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Contributions

J.M., Y.L. and R.J. conceived the project, designed and supervised the experiments, interpreted results and wrote the paper with input from R.M.R., and all other authors. P.A. provided primary AMN and ALD fibroblast cultures and helped with study inception and manuscript revision. L.-H.T. provided iPS-wt5 and input on study design and manuscript revision. J.M., Y.L. and R.J. designed the differentiation method and growth conditions for pMGLs. B.Y. performed transcriptome profile analyses and comparisons. M.M. provided additional hES and hiPS lines for the study and performed their pMGL differentiation. J.M. and Y.L. performed and analyzed all other experiments. A.O., G.B. and S.C. assisted with cell culture, sample preparation and data analysis.

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Correspondence to Rudolf Jaenisch.

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Competing interests

R. Jaenisch is an adviser to Stemgent, and cofounder of Fate Therapeutics and Fulcrum Therapeutics. R. Ransohoff is Senior Research Fellow with Biogen. P. Aubourg is cofounder of GTDesign.

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Muffat, J., Li, Y., Yuan, B. et al. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nat Med 22, 1358–1367 (2016). https://doi.org/10.1038/nm.4189

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