Differentiation of human and murine induced pluripotent stem cells to microglia-like cells

Abstract

Microglia are resident inflammatory cells of the CNS and have important roles in development, homeostasis and a variety of neurologic and psychiatric diseases. Difficulties in procuring human microglia have limited their study and hampered the clinical translation of microglia-based treatments shown to be effective in animal disease models. Here we report the differentiation of human induced pluripotent stem cells (iPSC) into microglia-like cells by exposure to defined factors and co-culture with astrocytes. These iPSC-derived microglia have the phenotype, gene expression profile and functional properties of brain-isolated microglia. Murine iPSC-derived microglia generated using a similar protocol have equivalent efficacy to primary brain-isolated microglia in treatment of murine syngeneic intracranial malignant gliomas. The ability to generate human microglia facilitates the further study of this important CNS cell type and raises the possibility of their use in personalized medicine applications.

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Figure 1: Human iPSCs are differentiated into microglia-like cells.
Figure 2: Phenotypic and genotypic analysis indicate human microglia-like cells.
Figure 3: Human iPS-MG demonstrate functional abilities consistent with human microglia.
Figure 4: Mouse iPSCs are differentiated into microglia using a two-stage process.
Figure 5: Mouse iPS-MG display phenotypic markers and gene expression consistent with primary neonatal microglia.
Figure 6: Mouse iPS-MG demonstrate functional abilities of neonatal microglia.
Figure 7: Murine nMG and iPS-MG prolong the survival of tumor-bearing animals.

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Acknowledgements

This work was supported by the Intramural Research Programs of the National Institute of Neurological Disorders and Stroke, the National Human Genome Research Institute and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

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Authors

Contributions

J.K.P. conceived the project; H.P., M.J.S., D.M.I., Y.C., D.B.M. and J.K.P. designed the experiments; H.P., M.J.S., D.M.I., Y.C., A.B.S., G.K. and M.A.B. performed the experiments; H.P., M.J.S., D.B.M. and J.K.P. analyzed the data; K.R.J. performed microarray data analysis; A.G.E., D.M., C.L.S. and S.G. provided technical support; H.L.M. provided conceptual advice; and H.P., M.J.S., D.B.M. and J.K.P. wrote the manuscript.

Corresponding authors

Correspondence to Dorian B McGavern or John K Park.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The stage 2 co-culture of human iPS-MG and the astrocyte feeder layers were analyzed by staining with ENTPD1 (CD39).

Co-cultures of human iPS-MG and the astrocyte feeder layers were analyzed by staining with ENTPD1 (CD39), a cell surface protein specific for microglia (right). Isotype antibody (left) was used as a control. Data is representative of two different differentiation experiments.

Supplementary Figure 2 Gene-expression data extracted from the human microarray for the six microglia-specific genes.

Our iMG show high expression of all the six microglia genes whereas the macrophages (Mac) and dendritic cells (DC) exhibit high expression of only selected genes. Data is representative of three different differentiation experiments.

Supplementary Figure 3 The stage 2 co-culture of murine iPS-MG and the astrocyte feeder layers were analyzed by GFP+ cell sorting.

The iPS-MG were differentiated from CX3CR1-GFP+ iPSC cells. The CX3CR1 gene is specifically expressed in microglia, and hence the iPS-MG express GFP and can be conveniently separated from the co-culture through FACs sorting. Data is representative of three different differentiation experiments.

Supplementary Figure 4 miPSC cells generated from Cx3cr1Gfp/+ knock-in mouse embryonic fibroblasts demonstrate properties of identity, authenticity and pluripotency.

Cx3cr1gfp/+ iPSCs stain for alkaline phosphatase (a), express the stem cell markers Oct4 (b) and Sox2 (c), form teratomas (d), can be used to generate chimeric mice (e), and display a normal karyotype (f). Data is representative of three different cell-culture for the alkaline phosphatase and immunocytochemistry experiments and 4 mice for the chimera experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1 (PDF 6827 kb)

Supplementary Methods Checklist (PDF 1093 kb)

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Pandya, H., Shen, M., Ichikawa, D. et al. Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nat Neurosci 20, 753–759 (2017). https://doi.org/10.1038/nn.4534

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