G CSF has a prominent effect on the neuronal differentiation of adult neural stem cells. We therefore determined whether GM CSF NSC639966 might have a similar role also on the Inhibitors,Modulators,Libraries differentia tion of stem cells. Results The GM CSF receptor alpha is expressed on adult neural stem cells The GM CSF receptor is expressed in Inhibitors,Modulators,Libraries a broad variety of brain regions, with a preferentially neuronal expression pattern. We also detected expression in regions where neural stem cells persist in the adult brain, such as the dentate gyrus. GM CSFR was expressed by cells in the subgranular zone, and in cells of the granular layer that are reminiscent of migrating stem cells. We also detected expression of the receptor on adult neural stem cells from the hippocampus, that also stained posi tive for the stem cell marker nestin.

Stem cells in culture also expressed the GM CSF receptor as judged by RT PCR. GM CSF promotes neuronal differentiation of adult neural stem cells We therefore asked whether GM CSF influenced the dif ferentiation of adult neural stem cells. To determine if GM CSF potentially influenced the generation of mature neural cell Inhibitors,Modulators,Libraries types we assayed the expression of the neuro nal marker genes III tubulin and neuron specific eno lase, the oligodendrocytic marker proteolipid protein, and the astrocytic marker glial fibrillary acidic protein by quantitative PCR in adult neural stem cells. 3 days after GM CSF treat ment of adult neural stem cells we detected a significant induction of III tubulin, and a non signifi cant elevation of NSE, whereas regulation of PLP or GFAP was not detectable.

To determine whether this induction of mature neuronal marker expression Inhibitors,Modulators,Libraries was concentration dependent, we assayed GM CSF induced differentiation by a luciferase reporter assay. Adult neural stem cells were transfected with the pGL3 p III tubulin reporter vector and stimu lated with increasing concentrations of GM Inhibitors,Modulators,Libraries CSF. We detected a concentration dependent activa tion of the III tubulin promoter after 48 h of GM CSF exposure. As positive control, NSCs were treated with the standard differentiation protocol involv ing withdrawal of EGF and bFGF, and addition of FCS. 10 ng ml GM CSF had the same induction potential com pared to this standard protocol. While the above assays measure marker induction on the cell population level, we used fluorescence activated cell counting to determine differentiation on the cellular level.

The percentage of MAP 2 positive cells doubled under GM CSF treatment. In summary, GM CSF promoted neuronal differentiation inhibitor expert of neural stem cells in vitro. Discussion Recently we uncovered that the hematopoietic factor GM CSF is also a neuronal growth factor with strong anti apoptotic actions on neurons. This characteristic is shared with two other important hematopoietic factors, EPO and G CSF.