Isolation and culture of adult neural stem cells (NSCs)
Adult rats were killed by decapitation. NSCs were isolated from the lateral subventricular zone using visually guided micro-preparation under a dissection microscope (Zeiss, Jena, Germany, magnification 8x [2]). About 3 mm of subventricular tissue was prepared with fresh scalpel blades, isolated with Dumont forceps and collected in ice-cold HBSS (Gibco, Eggenstein, Germany) containing 300 mg/ml D-glucose (Sigma, Deisenhofen, Germany). Cells from adjacent tissue not containing stem cells generally do not proliferate in growth-promoting medium. Isolated tissue was digested at 37°C with 1.33 mg/ml trypsin (Sigma, Deisenhofen, Germany), 0.7 mg/ml hyaluronidase (Sigma, Deisenhofen, Germany), 200 U/ml DNAse (Sigma, Deisenhofen, Germany) and 0.2 mg/ml kynurenic acid (Sigma, Deisenhofen, Germany) to dissociate cells. The tissue was passed through a 70 μm cell strainer (BD Falcon; Heidelberg, Germany) and transferred to ice-cold, serum-free medium containing BSA (Sigma, Deisenhofen, Germany) to stop trypsin activity. NSCs were cultured in serum-free medium containing basic fibroblast growth factor (bFGF; 10 ng/ml, Chemicon, Hofheim, Germany), epidermal growth factor (EGF; 20 ng/ml; R&D Systems, Wiesbaden, Germany) and B27 supplement (Gibco, Eggenstein, Germany). Primary neurospheres were dissociated at day 8–10 using Accutase (PAA, Pasching, Austria) to derive clonal neurospheres. The sub-culturing protocol consisted of neurosphere passaging every 3–4 days with whole culture medium change (fresh growth factors were added).
Immunocytochemistry
Neurospheres were harvested on microscope slides by cytospin centrifugation (212 g, 5 min., Shandon, Thermo, Dreieich, Germany), fixed in 3.7% PFA for 60 min at 4°C and washed 3x in 1x PBS for 5 min. Blocking was performed in 5% appropriate serum for 30 min followed by incubation with anti-Nestin (1:100, BD Pharmingen, Heidelberg, Germany); anti-GFAP (BD Pharmingen, Heidelberg, Germany, 1:100), anti-β-III-tubulin (Promega, Mannheim, Germany, 1:50), anti-TNF-RI (abcam, Cambridge, UK, 1:100), anti-TNF-RII (Alexis Biochemicals, Grünberg, Germany), anti-LeX (Developmental Hybridoma Bank, Iowa City, USA, 1:100), anti Sox2 (Sigma, Deisenhofen, Germany, 1:100), anti-Musashi (Chemicon, 1:100), anti-L1 (Developmental Hybridoma Bank, Iowa City, USA, 1:100), anti-PSA-NCAM (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-Notch1 (Developmental Hybridoma Bank, Iowa City, USA, 1:100), anti-Notch2 (Santa Cruz, 1:50), anti-A2B5 (Chemicon, 1:100), and anti DCX (Santa Cruz, 1:50), anti-TAK-1 (Santa Cruz, 1:50), anti IKK-β (Biosource, 1:50) and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, USA). Positive antibody binding was detected using a Cy3-conjugated antibody (1:300, Jackson Immuno Research Laboratories, distributed by Dianova, Hamburg, Germany). Nuclei were stained with SYTOX (1:10000, Molecular Probes, Göttingen, Germany). Staining was visualized using confocal laser scanning microscopy (LSM Pascal, Zeiss, Jena, Germany).
Reverse transcription polymerase chain reaction
Total RNA was isolated (RNAeasy, Qiagen, Hilden, Germany) from adult neurospheres according to the manufacturer's instruction and reverse transcribed. To exclude DNA contamination, mRNA was treated with DNAse (Qiagen). The PCR primers (Qiagen) were: 5'- ATGGGTCTCCCCATCGTGCCTG, 3'- TTATCGCGGGAGGTGGGTCGTG for TNF-RI and 5'- CACACAGTGCCCGCCAAGGTTGT, 3'-TCAAGGCACTTTGACAGCAATCTGGTC for TNF-RII.
Analysis of neurosphere growth
Secondary neurospheres were harvested, dissociated using Accutase (PAA, Pasching, Austria) and passed through a 40 μm cell strainer (BD Falcon, Heidelberg) to obtain a single neural stem cell suspension. Cells were cultured in serum-free medium containing basic fibroblast growth factor (bFGF; 20 ng/ml Chemicon, Hofheim, Germany), epidermal growth factor (EGF; 20 ng/ml; R&D Systems, Wiesbaden, Germany) and B27 supplement (Gibco, Eggenstein, Germany) and 10 ng/ml TNF (Calbiochem, Schwalbach, Germany). Control cells were cultured in neurosphere medium without TNF. The volume of living neurospheres was measured using an inverse microscope (Axiovert 100, Carl Zeiss, Jena, Germany) and ImageJ software (National Institute of Health, USA; [46]). Statistical significance was determined by ANOVA with Bonferroni correction, using GraphPad's Prism. P ≤ 0.05 was considered significant.
BrdU incorporation assay
BrdU assays were performed as described [47]. Briefly, 10 μM bromodeoxyuridin (BrdU, Sigma, Deisenhofen, Germany) was added to neurosphere cultures each day. Seventy-two hours after TNF stimulation (10 ng/ml), the neurospheres were harvested on microscope slides by Cytospin centrifugation (212 g, 5 min., Shandon, Thermo, Dreieich, Germany). The cells were fixed with 3.7% PFA for 60 min at 4°C and counterstained with 0.3 μM ToPro-3-iodite (Molecular Probes, Göttingen, Germany). Incorporation of BrdU into newly-synthesized DNA in the NSCs was detected by BrdU-dependent fluorescence enhancement of ToPro-3-iodite. Fluorescence was monitoring using an inverse confocal laser scanning microscope (LSM 5, Pascal, Carl Zeiss, Jena, Germany). Relative cell proliferation was calculated from the fluorescence of 5 fields of view (n = 4 for each condition) as follows: relative percentage of cell proliferatio = (Fexp - Fmin)/(Fmax - Fmin) × 100. Fexp is the fluorescence of the experimental test condition, Fmax is the maximal fluorescence and Fmin is the background fluorescence. Differences in relative cell proliferation were assessed by two-way ANOVA followed by a post-hoc t-test with Bonferroni correction. Differences between two conditions at P ≤ 0.05 were considered statistically significant.
Apoptosis assay
Neurospheres were prepared, cultured and collected as described above, with either 10 ng/ml TNF or neurosphere medium alone. After fixation, an In Situ Cell Death Detection Kit FITC (Roche, Mannheim, Germany) was used for immunocytochemical detection of apoptosis, based on labeling of DNA strand breaks (TUNEL technology) according to the manufacturer's instructions. Fluorescein isothiocyanate (FITC) fluorescence was monitored using an inverse confocal laser scanning microscope (LSM 5, Pascal, Carl Zeiss, Jena, Germany). As positive control, neurospheres were treated with DNAse (2000 U/ml, Sigma, Deisenhofen, Germany). As negative control, labeling solution containing FITC was used. The relative percentage of apoptotic cells was calculated from the fluorescence of 4 fields of view (n = 4 for each condition, relative percentage of apoptotic cells = (Fexp - Fmin)/(Fmax - Fmin) × 100; key as above). Statistical significance was determined by ANOVA. P ≤ 0.05 was considered significant.
Determination of cell number
Neural stem cells were plated at 1.0 × 106 cells/ml in triplicate for each condition. For pharmacological blockade of the NF-κB pathway, neural stem cells were pre-treated for 30 min with 0.1 mM pyrrolidine dithiocarbamate (PDTC, Sigma, Deisenhofen, Germany). TNF-α was added to the cultures at plating. Spheres were collected at 6, 24, 48 and 72 h after plating and dissociated, and total cell numbers were counted. Results were expressed as the mean ± SEM. Statistical significance was determined using two-way ANOVA followed by a post-hoc t-test with Bonferroni correction. Differences between two conditions at P = 0.05 were considered statistically significant.
Neuronal differentiation of TNF-treated neurosphere-derived cells
Secondary neurospheres were harvested and dissociated as described above. NSCs were cultured in serum-free medium containing B27 supplement and TNF but without EGF and bFGF or in medium without TNF addition and plated on poly-D-lysine/laminin-coated culture slides (BD Biocoat, Heidelberg, Germany). Three days after the TNF stimulus the cells were fixed, stained and analyzed as described above.
Glial differentiation
Secondary neurospheres were harvested and dissociated, followed by culture in DMEM/F:12 (Gibco) and 10% FBS (PAA, Pasching, Austria). After 4 days of culture the cells were fixed, stained and analyzed as described above.
Reporter gene assay (transfection and analysis)
Neurospheres were dissociated as described above and transfected using a Rat NSC Nucleofector Kit (Amaxa, Köln, Germany) according to the manufacturer's instructions with minor modifications. In particular, 5.0 × 106 cells were used for each transfection. After dissociation of the neurospheres (see above), cells were centrifuged at 210 × g for 10 min and re-suspended in an appropriate volume of Amaxa Nucleofector solution. After addition of DNA, the cells were electroporated in an Amaxa device and collected in 10 ml preheated cytokine-free media. After further centrifugation at 210 × g (10min), the cells were re-suspended at appropriate density in cytokine-containing medium. Transfection efficiency was measured using the pmaxGFP vector (Amaxa) and analysis by fluorescence microscopy (Axiovert 100, Carl Zeiss, Jena) and flow cytometry (FACScalibur, Becton Dickinson, Heidelberg, Germany).
To detect NF-κB activity in TNF-stimulated versus unstimulated neural stem cells, a Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) and κB-luc Reporter (BD Clonetech, Heidelberg, Germany) was used. All cells were transfected with κB-luc reporter vector and Renilla-luc control vector (Promega, Mannheim, Germany). IκB-AA1 (super-repressor of NF-κB, cloned into the commercially available Rc/CMV expression vector (Promega) [48]) was cotransfected. As a mock vector, pMETalpha (Invitrogen) without insert was used. Forty-eight hours after transfection, the cells were lysed and assayed for promoter-dependent luciferase activity versus promoter independent Renilla-luc activity (Lumat LB9507 device, Berthold Technologies, Bad Wildbach, Germany). A representative experiment is shown.
Detection of nuclear NF-κB in TNF-stimulated neural stem cells
Neurospheres were stimulated with 10 ng/ml TNF. Thirty minutes after the TNF stimulus, the cells were harvested and fixed as described above. After 30 min permeabilization with 0.5% TritonX100/PBS and blocking with 5% goat serum, the cells were stained with anti-p65 antibody (Chemicon, Hofheim, Germany, 1:50). Nuclear NF-κB was detected with Cy3-conjugated anti-mouse antibody (1:300, Jackson Immuno Research Laboratories, distributed by Dianova, Hamburg, Germany). Nuclei were stained with SYTOX (1:10000, Molecular Probes, Göttingen, Germany). Antibody staining was visualized using confocal laser scanning microscopy (LSM Pascal, Zeiss, Jena, Germany). Laser power and detector settings were kept constant to maintain consistency in the data collection system. Fluorescence intensity was quantified using Image J image analysis software [46]. Statistical significance was determined by ANOVA with Bonferroni correction, using GraphPad's Prism. P < 0.05 was considered significant.
Detection of TNF-induced cyclin expression
Neural stem cells were dissociated, cultured for 24 h as described above and synchronized by cold shock (24 h, 4°C). After 4 h equilibration at 37°C (release into cell cycle), 10 ng/ml TNF-α was added.
Four hours after TNF stimulation, neurospheres were harvested on microscope slides by Cytospin centrifugation (212 g, 5 min, Shandon, Thermo, Dreieich, Germany), fixed in 3.7% PFA for 60 min at 4°C and washed 3x in 1x PBS for 5 min followed by permeabilization with 0.1% Triton X-100 for 30 min. Blocking was done in 5% appropriate serum for 30 min followed by incubation with anti-cyclin D1 (Sigma, Deisenhofen, Germany, 1:100), anti-cyclin D2 (abcam, Cambridge, UK 1:100) and anti-cyclin D3 (Sigma, Deisenhofen, Germany, 1:100). Antibody binding was detected with Cy3-conjugated antibody (1:300, Jackson Immuno Research Laboratories, distributed by Dianova, Hamburg, Germany). Nuclei were stained with SYTOX (1:10000, Molecular Probes, Göttingen, Germany). Antibody staining was visualized using confocal laser scanning microscopy (LSM Pascal, Zeiss, Jena, Germany).
Gene silencing (siRNA design and transfection)
The Ambion Silencer siRNA Construction Kit (Ambion) was used to produce siRNAs against TAK-1 and IKK-β. The target sequences were identified and appropriate oligonucleotides constructed as per the manufacturer's instructions. The target sequences for TAK-1 and IKK-β knock down were identified, followed by Blast searches to ensure that the sequences did not contain significant homology to any other known genes. The sequences were TGGCTTATCTTACACTGGA for TAK-1 and GGTGGAAGAGGTGGTGAGC for IKK-β. Both produced similar levels of specific gene product knockdown. In addition, siRNA for GFP (Amaxa, Köln, Germany) was used as a control.
Cells were transiently co-transfected as described above, either with siRNA for the TAK-1 and CMV driven GFP or with siRNA for IKK-β and CMV-GFP, to monitor the efficacy of transfection. All cells were counterstained with DAPI. As control, anti-GFP siRNA and CMV-GFP were transfected. After 48 h, the down-regulation of TAK-1 and IKK-β was analyzed using fluorescence microscopy (Axiovert, Zeiss, Jena, Germany) and western blots.
Western blot analysis
Soluble cell protein (250 μg per condition) was separated by SDS-PAGE using 8% polyacrylamide gels and electroblotted on to PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked in TBST+3% nonfat dry milk. The following first antibodies were used: mouse monoclonal anti-IKK-β (Biosource) and goat polyclonal anti-TAK-1 (Santa Cruz Biotechnology). For detection, horseradish peroxidase-conjugated goat anti-mouse (Bio-Rad Laboratories GmbH, Muenchen, Germany) and donkey anti-goat (Santa Cruz Biotechnology) IgGs were used with an ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Influence of gene silencing on proliferation, NF-κB activity, cyclin D1 expression and apoptosis
Anti TAK-1 and anti IKK-β siRNA transfected adult neural stem cells were cultivated for 24 h. Cells were then stimulated with 10 ng/ml TNF and cultivated for up to 48 h. The total cell number was determined as described above.
To investigate NF-κB activity, the cells were co-transfected with siRNA for TAK-1 and κB-luc reporter vector or siRNA for IKK-β and κB-luc. Twenty-four hours after transfection, the cells were stimulated with 10 ng/ml TNF and cultivated for an additional 24 h, then lysed and assayed for promoter-dependent luciferase activity as described above. To study the influence of gene product knock down on cyclin D1 expression and apoptosis, the cells were transfected with siRNAs for TAK-1 and IKK-β followed by 24 h cultivation. Cells were then stimulated with TNF, cultured for an additional 24 h, stained for cyclin D1 and cleaved caspase3 (Cell Signaling Technologies, Danvers, USA, 1:100) and counterstained with DAPI. GFP-positive cells were analyzed for TAK-1 and IKK-β immunoreactivity.