We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures.

Cells were harvested by centrifugation and proteins were extracted for 60 min at room temperature in a lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) Triton X-100, 100 mM DTT, 0.05% IPG buffer pH 3–10 (GE Healthcare, Amersham Biosciences, Uppsala, Sweden), and 0.156% (w/v) Complete protease inhibitor tablets (Roche, Mannheim, Germany). Sample protein amounts were determined by the Bradford method [24]. Two-dimensional gel elctrophoresis was performed according to [5,22,25]. Briefly, 5–10 μL of the protein solution were suspended in rehydration solution consisting of 6 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3–10, and a few grains of bromophenole blue in a final volume of 350 μL. The samples were applied to 18 cm pH 3–10 non-linear gradient IEF gel strips for isoelectric focussing the IPGphor apparatus (GE Healthcare, Amersham Biosciences, Uppsala, Sweden). The IEF gel strips were allowed to reswell for 12 hours at 30 V. Then 200 V, 500 V and 1000 V were applied for 1 hour each. Voltage was increased to 8000 V in 30 min and kept constant at 8000 V for 12 hours, resulting in a total of 100,300 Vh. Gel strips were equilibrated for 20 min each in an SDS equilibration buffer consisting of 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a few grains of bromophenole blue, and 1% (w/v) dithiothreitol or 2.5% (w/v) iodoacetamide, respectively. The second dimension separation was performed using 12.5% polyacrylamide gels in the presence of 0.1% (w/v) sodium dodecylsulfate. The gels were run at 30 mA for 30 min and 100 mA for about 4 h in a 20 cm × 20 cm water-cooled vertical electrophoresis apparatus (OWL, Woburn, MA, USA). For image analysis, gels were silver stained [26]. Briefly, gels were fixed over-night in 40% (v/v) methanol containing 10% (v/v) acetone. After 3 times of washing in 30% (v/v) ethanol for 20 min each, gels were impregnated in 0.02% (w/v) sodium thiosulfate. Then the gels were washed quickly in bidestilled water and stained with 0.2% (w/v) silver nitrate for 20 min. Again the gels were washed quickly in bidestilled water and carefully developed in 3% (w/v) sodium carbonate, 0.05% (v/v) formole 37%, 0.0005% (w/v) sodium thiosulfate. After washing 3 times with bidestilled water, the reaction was stopped by 0.5% (w/v) glycine.

Gels were scanned and images were analyzed by the Phoretix 2D Advanced software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK). Statistical analysis of the spot volumes defined as integral of spot area multiplied by optical density was done comparing normalized means ± standard deviations from 5 gels of each group by Student's t-test for unpaired data [27]. Three-dimensional reconstruction of protein spot volumes was done as reported [5]. Spots of interest were excised and digested by trypsin for mass spectrometry. Peptide mass fingerprinting (PMF) using MALDI-TOF, peptide sequencing using LIFT-TOF/TOF, data acquisition, and database search was performed by the mass spectrometry group at ProteoSys (Mainz, Germany) using an Ultraflex TOF/TOF (Bruker-Daltonics, Bremen, Germany) as described priviously [27]. Mass spectra were identified by searching the NCBI non-redundant protein database using Mascot [28,29].

Several drops of cell culture medium containing neurospheres and single cells either from subventricular zone or olfactory bulb were placed on a glass slide, sealed with a cover slip and frozen at -80°C. For staining, the cover slip was removed quickly and the cells on the slide were fixed for 10 min in 4% paraformaldehyde in PBS, pH 7.4, with 0.2% Tween-20 (PBST). Slides were blocked 1 h at room temperature in 5% milk powder in PBST and incubated with antibodies against the neural progenitor marker nestin (1:100, BD Biosciences, Heidelberg, Germany), the astrocytic marker GFAP (1:100; BD Biosciences, Heidelberg, Germany), the neuronal markers tubulin-beta-III (1:500; clone TuJ-1; Abcam, Cambridge, UK), microtubulus-associated protein MAP2b (1:100; Chemicon, Cambridge, UK), and Glucose Transporter GLUT3 (1:250; Biogenesis, Newcastle-upon-Tyne, UK) overnight at 4°C in a humidified chamber. The next day, slides were washed 3 times for 5 min in PBST and incubated with a suitable Cy3- or Cy5-conjugated secondary antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 3 h at room temperature. After washing 3 times for 5 min each with PBST, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, 1:5,000, Molecular Probes, Eugene, OR, USA) for 5 min. Slides were embedded in fluoprotective mounting media [30] and stored at 4°C in the dark until fluorescence microscopy was performed.