Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells.

INTRODUCTION: Studying primary adult microglia is hampered because of the difficult isolation procedure and the low cell yield. We therefore established a differentiation protocol using a culture system developed for the expansion of non-adherent bone marrow cells. METHODS: Non-adherent bone marrow derived stem cells (NA-BMC) are derived by selective adhesion ('preplating') and are non adhesive adult stem cells. We investigated the changes in bone marrow cell populations by this repeated selective adhesion and compared the potential of the derived cells to differentiate towards microglia. Cells were differentiated with astrocyte conditioned medium (ACM) and granulocyte-monocyte colony stimulating factor (GM-CSF). RESULTS: NA-BMC cultures show a steep raise in the fraction of stem cells during the cultivation time and the differentiation potential is of the same quality as established protocols. Around 70% of the cells are microglia defined as being positive for CD11b/CD45 and show phagocytosis activity and oxidative bursts. CONCLUSION: The non-adherent cell system has the advantage that is produces stem cell progenitors during expansion and provides good microglial differentiation.

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

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.