Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

Abstract

Background: Microglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM) stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L), astrocyte-conditioned medium (ACM) and GM-CSF on the differentiation to microglia-like cells.

Methods: We assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80), but also for the first time for functional performance (phagocytosis, oxidative burst) and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices.

Results: The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation.

Conclusion: We conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.

Figure 1

Flow cytometric analysis of adherent? BM cells. (A) Flow cytometric analysis of adherent? BM cells on CD11b/CD45 expression (n = 3) following culture in vitro for certain period of time. Representative scatter plots of CD11b/CD45 labeled differentiated cells and primary microglia, fresh bone marrow and an isotype control. (B) Flow cytometric analysis of adherent? BM cells on their F4/80 expression (n = 3). Representative histogram plots of F4/80 labeled differentiated cells, primary microglia and fresh bone marrow. Isotype control gray, F4/80 labeled cells black. *** = P < 0.001, ** = P < 0.01, * = P < 0.05.

Figure 2

Time course of CD11b/CD45 and F4/80 expression of untreated and cytokine treated BMC (n = 3). Time course of CD11b and F4/80 medians. Significant changes are denoted with respect to freshly isolated BMC on day 0. *** = P < 0.001, ** = P < 0.01, * = P < 0.05.

Figure 3

Phagocytosis and Oxidative burst of differentiated cells. (A) Phagocytosis of differentiated cells and fluorescence microscope picture of phagocytosis of non adherent BM cells differentiated with ACM/GM-CSF (n = 3). Arrows indicate the same cell in bright field and fluorescence picture. Fluorescence images were taken with a Zeiss Axio Observer at 200× (left) and 400× (right) magnification. (B) Oxidative burst of differentiated cells, representative histogram plot of inactive (open histogram) and PMA activated (red histogram) cells (n = 3). The shift between un-treated and PMA treated microglia was measured as quotient between medians of treated and untreated cells (Fluorescence signal noise ratio - FSN). Cells with no shift are based at 1 and higher numbers represent cell populations which did show ROS production. Light microscope picture of NBT reduction of non adherent BM cells supplemented with ACM/GM-CSF. Arrows indicate one cell with dark blue NBT precipitate and one cell without precipitate. The picture was taken with a Leica DM IL at 20× magnification. *** = P < 0.001, ** = P < 0.01, * = P < 0.05.

Figure 4

Representative light microscope pictures of differentiated cells. Images were taken with a Leica DM IL at 200× magnification. BMC: Whole bone marrow cells. Fluorescence picture of an Iba-1 stained microglia in a brain slice (B) was taken with an Axio Imager A1 (Zeiss) at 63× magnification.

Figure 5

Coculture with living brain slices. Differentiated ACM/GM-CSF treated BMC were labeled with DIO and seeded on brain slices on day 9. Counterstaining with propidium iodide was used to assess cell survival. After 1, 2, 3, 6 and 10 days slices were scanned with a Leica Microsystems TCS SP2 confocal microscope to assess survival of seeded cells and their migration into the tissue. Arrows indicate single cells that have already migrated through the surface after 2 days. Magnification was 100x and scanning depth was 160 μm.

Figure 6

Differentiated cells of all protocols were labeled with DIO and seeded on brain slices on day 9. After 10 days coculture slices were propidium iodide stained and scanned with a Leica Microsystems TCS SP2 confocal microscope to measure cell survival and their migration. Arrows indicate several cells that have migrated deeper into the brain tissue. The images were taken at 100× magnification and the brain slices were scanned to a depth of 160 μm.