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. 2014 Oct;40(6):697-713.
doi: 10.1111/nan.12086.

Development of a culture system to induce microglia-like cells from haematopoietic cells

Daisuke Noto  1 , Hiroshi Sakuma, Kazuya Takahashi, Reiko Saika, Ryoko Saga, Masahito Yamada, Takashi Yamamura, Sachiko Miyake
Affiliations
Free PMC article

Development of a culture system to induce microglia-like cells from haematopoietic cells

Daisuke Noto et al. Neuropathol Appl Neurobiol. 2014 Oct.
Free PMC article

Abstract

Aims: Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases.

Methods: To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay.

Results: We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell-cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses.

Conclusion: Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells.

Keywords: astrocytes; haematopoietic cells; interleukin 34; microglia; monocytes; triggering receptor expressing on myeloid cells-2 (TREM2).

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Figures

Figure 1
Figure 1
LN− cells cocultured with astrocytes. (A) Immunohistology of LN− cells derived from GFP mice cocultured with astrocytes. Cultures were stained with primary anti-CD11b, anti-TREM2 and anti-MHC II (IAb) antibodies followed by rhodamine-conjugated secondary antibody and DAPI. Arrows indicate GFP-positive large flat cells. Arrowheads indicate GFP-positive small round cells. Bar: 50 μm. (B) The percentage of round cells or flat cells among GFP+ or staining-positive cells was quantified by microscopic analysis. Data are presented as mean ± standard deviation (SD). (C) The percentage of CD11b-, TREM2- or MHC II-positive cells among GFP+ round or flat cells was quantified by microscopic analysis. Data are presented as mean ± SD. (D) Z-stack immunofluorescence confocal microscopy of coculture of GFP-positive LN− cells and astrocytes stained with anti-GFAP (red) and DAPI (blue). Bar: 10 μm. Data are presented as mean ± SD. *P < 0.05. Data are representative of three independent experiments.
Figure 2
Figure 2
Murine monocytes cocultured with astrocytes. (A) Fluorescent microscopic images of GFP+ LN− cells, GR-1+ monocytes or Gr-1− monocytes cocultured with astrocytes. Bar: 200 μm. (B) The numbers of GFP+ cells in A were quantified by microscopic analysis. Data are presented as mean ± standard deviation (SD). (C) Immunocytology of Gr-1+ monocytes derived from GFP mice cocultured with astrocytes. Cultures were stained with primary anti-CD11b, anti-TREM2 and anti-MHC II antibodies followed by rhodamine-conjugated secondary antibody and DAPI. Arrows indicate GFP+ large flat cells. Arrowheads indicate GFP+ small round cells. Bar: 50 μm. (D) The percentage of round cells or flat cells among GFP+ or staining-positive cells was quantified by microscopic analysis. Data are presented as mean ± SD. (E) The percentage of CD11b, TREM2- or MHC II-positive cells among GFP-positive round or flat cells was quantified by microscopic analysis. Data are presented as mean ± SD (F) Z-stack immunofluorescence confocal microscopy of coculture of GFP+ Gr-1+ monocytes and astrocytes stained with anti-GFAP (red) and DAPI (blue). Bar: 10 μm. Data are presented as mean ± SD. *P < 0.05. Data are representative of three independent experiments.
Figure 3
Figure 3
Cell surface markers of spleen monocyte-derived ML cells. (A) Immunocytology of spleen monocytes derived from GFP mice cocultured with astrocytes. Cultures were stained with primary anti-CD11b, anti-TREM2 and anti-MHC II antibodies followed by rhodamine-conjugated secondary antibody and DAPI. Arrows indicate GFP+ large flat cells. Arrowheads indicate GFP+ small round cells. Bar: 50 μm. (B) Flow cytometry analysis of brain microglia, LN− cell-derived ML cells, spleen monocyte-derived ML cells and splenic macrophages. Black histograms, staining with antibodies to markers below plots; grey histograms, isotype-matched control antibody. Data are representative of three independent experiments.
Figure 4
Figure 4
Morphological analysis of ML cells in brain slice cultures. (A) Confocal microscopic images of LN− cell-derived ML cells, LN− cells, monocyte-derived ML cells, monocytes, cultured microglia or spleen macrophages induced in brain slice cultures. Bar: 200 μm. (B) The ramification indexes of brain slice culture-induced cells were quantified by confocal microscopic analysis. Data are presented as mean ± SD. *P < 0.05.
Figure 5
Figure 5
Human monocytes cocultured with astrocytes. (A) Immunocytology of human monocytes cocultured with astrocytes. Cultures were stained with primary anti-CD11b, anti-TREM2 and anti-GFAP antibodies followed by FITC- or rhodamine-conjugated secondary antibody and DAPI. Bar: 50 μm. (B) The number of monocyte-derived cells was quantified by microscopic analysis. Data are presented as mean ± standard deviation (SD). (C) Flow cytometry analysis of monocytes and monocyte-derived ML cells. Black histograms, staining with antibodies to markers below plots; grey histograms, isotype-matched control antibody. Data are representative of three independent experiments. (D) Z-stack immunofluorescence confocal microscopy of coculture of monocytes and astrocytes stained with anti-GFAP (green), anti-TREM2 (red) and DAPI (blue). Data are representative of three independent experiments. Bar: 10 μm.
Figure 6
Figure 6
Differentiation assays of LN− cells. (A) Immunocytology of mouse LN− cells cultured on murine astrocytes with or without M-CSF or IL-34. Bar: 50 μm. (B) Dose–response relationship between M-CSF/IL-34 and the number of ML cells differentiated from LN− cells (cell number per mm2). *P < 0.05 as compared with untreated cells. (C) Quantitative analysis of small round-shaped cells and large flat cells differentiated from LN− cells (cell number per mm2). (D) Morphological characteristics of LN− cells differentiated with M-CSF or IL-34. Bar: 10 μm. (E, F) Quantitative analysis (E) and immunocytology (F) of mouse monocytes cultured on murine astrocytes in the absence or presence of M-CSF or IL-34, Bar: 50 μm. (G, H) Fluorescent microscopic images (G) and quantitative analysis (H) of LN− cells cultured on astrocytes with or without anti-IL-34 antibody. IL-34 neutralization resulted in the significant reduction of round cells. *P < 0.05 as compared with cells treated by control IgG. Bar: 50 μm. (I) Intracellular staining of IL-34 of mouse primary mixed glial cells. Some GFAP-positive astrocytes showed granular staining in cytoplasm (arrowhead), whereas other astrocytes were not stained with anti-IL-34 Ab (arrow), Bar: 20 μm. Data are presented as mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.00005.
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