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Updated: Aug 19, 2026
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Characterization of microglia induced from mouse embryonic stem cells and their migration into the brain parenchyma
Takahiro Tsuchiya1, Kae Chang Park, Shinichi Toyonaga
1Department of Neurosurgery, Kochi Medical School, Kochi University, Kohasu, Okoh-cho, Nankoku, Kochi 783-8505, Japan.
Abstract:
We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1(+)/CD45(low) and Mac1(+)/CD45(high) to define microglia and macrophages, respectively, we show that Mac1(+) cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45(low) expression was high and CD45(high) expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1(+) cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-gammaR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-gamma. Intravenously transferred GFP(+) microglia derived from GFP(+) ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP(+) cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP(+) and Iba1(+) cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.
Insights
Mouse embryonic stem cells can be differentiated into functional microglia-like cells. These cells selectively migrate to the brain after intravenous transfer, offering a promising tool for neurological research.
Area of Science:
- Neuroscience
- Stem Cell Biology
- Immunology
Background:
- Microglia play crucial roles in brain function and disease.
- Deriving microglia from stem cells is challenging but holds therapeutic potential.
Purpose of the Study:
- To develop a protocol for deriving functional microglia-like cells from mouse embryonic stem cells (ES cells).
- To assess the characteristics and in vivo behavior of these derived microglia.
Main Methods:
- Mouse ES cells were differentiated using a five-step method with GM-CSF stimulation.
- Density gradient centrifugation was employed to isolate microglia-like cells.
- Flow cytometry and immunofluorescence were used to characterize cell markers (Mac1, CD45, Iba1) and gene expression.
- GFP-labeled ES cell-derived microglia were intravenously transplanted into mice to track their distribution.
Main Results:
- A high yield of microglia-like cells (approx. 90% Mac1+) was obtained.
- These cells expressed key microglial markers (MHC class I/II, CD40, CD80, CD86, IFN-gammaR).
- Transplanted GFP+ microglia selectively migrated to the brain (corpus callosum, hippocampus) and exhibited mature ramified morphology.
Conclusions:
- The developed protocol effectively generates functional microglia-like cells from mouse ES cells.
- These cells demonstrate selective brain homing and possess characteristics of mature microglia.
- This provides a valuable in vitro and in vivo model for studying microglia and potential cell-based therapies.
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