Background: Microglia are the phagocytes of the brain. They have been implicated in the underlying causes of several degenerative diseases and display a loss of function and loss of balanced regulation during age. Microglia are thought to originate from special progenitor cells during embryogenesis and from myeloid progenitor cells migrating from bone marrow to the brain. Throughout life microglia are replenished at a slow rate both by limited proliferation of resident microglia and by the continuous migration of bone marrow derived progenitor cells.
We investigated the feasibility to differentiate microglia from non adherent bone marrow cells (NA-BMC). Furthermore we looked at the effects of Flt3, SCF and GMCSF and we compared and adapted several protocols for differentiation to microglia. We assessed microglia differentiation not only by marker expression but also by functional performance and tested the migration and integration into living brain tissue.
Materials and Methods: Bone marrow cells were differentiated to microglia employing various combinations of selective adhesion and astrocyte conditioned medium (ACM), Flt3, SCF and GMCSF supplementation. Uptake of fluorescent beads, burst in ROS production and microglia markers (CD11b/CD45, F4/80) were quantified using flow cytometry. Migration was assessed with confocal microscopy in living brain slice cultures.
Results: The cells differentiated from bone marrow show function (phagocytosis, oxidative burst) and markers (CD11b+/CD45+, F4/80+) of primary microglia and they migrate into living brain tissue.
Conclusion: Several protocols employed yield high purity of cells which are phenotypic and functional equals of primary microglia. Differentiation from NA-BMC yields functional microglia. Flt3 shows both negative and positive effects on differentiation while GMCSF enhances, SCF hinders differentiation. In our dense bone marrow cultures in vitro culture time has a strong impact on differentiation.