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Generation of microglial cells from mouse embryonic stem cells

Nature Protocols volume 5, pages 1481–1494 (2010)Cite this article

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Abstract

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of ES cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of ES cell-derived microglial precursors (ESdMs). The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

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Figure 1: Flow chart of the protocol (Steps 15–57) with bright-field microscopic images.
Figure 2: Immunocytochemistry of cells during neural and microglial differentiation (Steps 37–56).
Figure 3: Analysis of ESdM surface markers.
Figure 4: Functional analysis of ESdMs.

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Acknowledgements

The Neural Regeneration Group at the University of Bonn Life & Brain Center is supported by the Hertie Foundation, the Walter und Ilse Rose Foundation, the Deutsche Forschungsgemeinschaft (FOR1336, KFO177, SFB704) and the EU (LSHM-CT-2005-018637). We thank J. Schumacher and R. Hass for excellent technical support of cultures and molecular biology.

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  1. Neural Regeneration Group, Institute of Reconstructive Neurobiology, University of Bonn, Bonn, Germany

    Clara Beutner, Kristin Roy, Bettina Linnartz, Isabella Napoli & Harald Neumann

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  1. Clara Beutner
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  2. Kristin Roy
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  3. Bettina Linnartz
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  4. Isabella Napoli
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  5. Harald Neumann
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Contributions

C.B. and H.N. conceived and designed the experiments. C.B. performed generation of ESdMs and did most of the characterization of the cells by immunocytochemistry, flow cytometry and qRT-PCR. K.R. carried out the phagocytosis assay and parts of the characterization by flow cytometry. B.L. defined the purity of the cell population and analyzed the migratory capacity of the cells. C.B., K.R., B.L. and H.N. wrote the paper. I.N. participated in the establishment of the generation protocol of ESdMs from ES cells and corrected the paper.

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Correspondence to Harald Neumann.

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The authors declare no competing financial interests.

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Beutner, C., Roy, K., Linnartz, B. et al. Generation of microglial cells from mouse embryonic stem cells. Nat Protoc 5, 1481–1494 (2010). https://doi.org/10.1038/nprot.2010.90

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