The G-CSF receptor is present on adult neural stem cells (NSCs) and drives neural progenitor differentiation in vitro. (A) Results of PCR analysis for the G-CSF receptor on neural stem cells demonstrate presence of G-CSF receptor mRNA in neurospheres in culture. (B) Double-fluorescence immunocytochemistry on neural stem cells plated onto coated 96-well plates. Almost all cells were positive for the stem cell marker nestin and for the G-CSF receptor (original magnification, ×20). (C–E) G-CSF drives neuronal phenotype induction in adult neural stem cells in vitro. (C) As 1 approach, we assayed β-III-tubulin promoter activity by a luciferase reporter assay. Treatment with G-CSF in increasing concentrations for 48 hours increased promoter activity. As a positive control, neural stem cells were treated with the standard differentiation protocol involving withdrawal of EGF and bFGF and addition of FCS (+FCS/–EGF/–bFGF). (D) G-CSF treatment induces a concentration-dependent upregulation of the neuronal markers NSE and β-III-tubulin 4 days after G-CSF treatment, as indicated by quantitative PCR. The glial markers PLP and GFAP are moderately induced in response to increasing G-CSF concentrations. Error bars indicate standard deviations calculated from measurements done with serial dilutions of the cDNA samples (1:3, 1:9, 1:27, and 1:81). (E) FACS analysis for MAP2-positive cells served to confirm the data presented above on an individual cell basis. Cells were treated for 4 days prior to analysis. Left: Examples of FACS runs of vehicle-treated and G-CSF–treated (100 ng/ml) neural stem cells. Right: The bar graph summarizes the results of several experiments (vehicle, n = 4; G-CSF, n = 7; P < 0.005).