The HMF conditions
The HMF condition for cell culture was established as described (Mo et al. 2013). The samples exposed to the HMF were cultured in a magnetic shielding chamber with a residual magnetic field <200 nT. The GMF control samples were cultured on a plastic shelf outside the magnetic shielding box with a local magnetic field of 15.1 ± 2.2 μT. The other conditions inside and outside the chamber were almost identical.
The HMF environment for animal rearing was provided by a 3-axis Helmholtz coils system (HCS) as described (Mo et al. 2015). The HMF-exposed animals were reared in a residual magnetic field of 0.029 ± 0.029 μT (center) and 0.55 ± 0.3 μT (average value); the control animals were reared on a wooden table (49.88 ± 1.82 μT average value), 1.5 m away from the HCS, in the same room (Table S1). The alternating magnetic fields (AMFs) were measured using a CCG-1000 induction alternative magnetometer (National Institute of Metrology, Beijing, China) and the predominant AMF frequencies were checked from the output of signal using a Textronics TDS 2014 digital real-time oscilloscope (Tequipment.NET, Long Branch, NJ, USA). The AMFs of the HCS and the GMF control environments were identical.
C57BL/6 mice were provided by the animal experiment center of the Institute of Biophysics (IBP). P2 (male/female), P15 (male), and 2-month (male) littermate naïve mice were used for primary NS cultures. 4–6 week male mice were used for the in vivo assay. All animal experiments were approved by the Animal Care and Use Committee at the IBP, Chinese Academy of Sciences (CAS) (authorized No.: SYXK2014-31) and carried out in accordance with the national guidelines for the care and use of laboratory animals.
For the HMF/GMF exposure assay, animals were firstly reared on the GMF for 7 days to adapt to the environment, and then randomly grouped as four mice per standard “shoebox” cage. The cages in the HMF group were aligned as described (Prato et al. 2005). Animals were reared under a 12 h/12 h light/dark cycle. Daily magnetic field fluctuation was recorded, and the room temperature and humidity was maintained at 22 ± 1°C and ~40%–60%, respectively (Fig. S4).
Primary neurosphere (NS) culture
The whole brain, SVZ and hippocampus of mice were obtained as described previously (Walker et al. 2008). Whole brain P2 mouse or adult SVZ samples were enzymatically digested (0.25% trypsin and 0.025% EDTA in PBS) for 7–10 min at 37°C, and the hippocampus were digested using papain [1 mg/mL papain (Sigma-Aldrich, St. Louis, MO) and 0.5 mg/mL DNase I in L-15 medium (Invitrogen/molecular probe, Grand Island, NY)] for 10–15 min at 37°C. The mixtures were then mechanically triturated and filtered through 40 μm sieves (BD Bioscience, San Jose, CA). Cells were collected by centrifugation and resuspended in NS culture media (NSA) [DMEM/F12 (Invitrogen) supplemented with 10% proliferation supplement (Stem Cell Technologies, Vancouver, British Columbia, Canada), 2% bovine serum albumin (BSA) (Roche, Basel, Switzerland), 2 μg/mL heparin (Sigma-Aldrich), 10 ng/mL fibroblast growth factor 2 (bFGF) (Roche), and 20 ng/mL epidermal growth factor (EGF) (BD Bioscience)]. Then the primary cells were plated in a 96-well plate (1000 cells/well with 200 μL NSA medium) for NS counting and size analysis, or in a 60 mm dish (8.0 × 105 cells/dish with 4 mL NSA medium) for cell counting. The number and size of mature NSs (diameter ≥ 40 μm) were determined with an inverted microscope.
The cultured NSs were collected and passaged at day 7. The NSs were washed with PBS, trypsinized and then mechanically triturated into dissociated cells in NSA medium. After cell counting (Ntotal d7), the cells were seeded at a density of 1000 cells per well with 200 μL NSA medium in a 96-well plate for NS counting and size analysis, or 1.0 × 105 cells (Nseed d0) per 60 mm dish with 4 mL NSA medium for counting total cell numbers (Ntotal d7). Cell proliferation rate (R) was calculated as: R = Ntotal d7/Nseed d0. The theoretical total number of cells obtained at a certain passage (NPi) was calculated as: NPi = Nseed P1*RP1*RP2*…*RPi.
For single clone culture, individual large NS (diameter ≥ 150 μm) was collected and trypsinized, as described above. All cells were seeded in a 6-well plate with 2 mL NSA medium per well. Successful cultures were determined when large NSs (diameter ≥ 150 μm) were observed at day 7. At least six NSs were used in each trial.
Cell division in the primary NS was measured with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) (Sigma-Aldrich) staining as described (Quah and Parish 2010). Primary cells from P2 mouse brain were stained with 25 μmol/L CFSE (107 cells/mL) for 20 min at 37°C. After two washes with PBS (with 0.1% BSA), CFSE-stained cells were seeded in a 60 mm dish and incubated in the HMF or GMF condition, as described above. NSs were collected and trypsinized at day 7, CFSE fluorescence was measured using a FACS Calibur flowcytometer (BD Bioscience) and analyzed with the Cell Quest Pro software. The cells that were not stained with CFSE were used as blank control (Blank).
Quantitative real-time PCR (qPCR)
Total RNA of primary NSs was extracted using an RNA extraction kit (QIAGEN, Hilden, Germany). cDNA samples were synthesized using an EasyScript First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). The gene-specific primers (Supplementary Table S2) were designed by PrimerBank (Wang et al. 2012). A TransStart Green qPCR SuperMix UDG kit (TransGen Biotech) was applied to prepare the qPCR samples, which were run in triplicate on a Rotorgen Q real-time PCR cycler (QIAGEN). Thermal cycling was performed at an initial UDG incubation step at 50°C and a UDG inactivation step at 94°C, and then subjected to 45 cycles of 15 s denaturing at 95°C, 30 s at annealing temperature, and a 30 s extension at 72°C. Quantitative gene expressions were referenced to Tubb5 and normalized to the GMF samples.
NS differentiation assay
NS differentiation was performed as described (Golmohammadi et al. 2008) under the GMF condition. Coverslips were pre-coated with 15% poly-ornithine (Sigma-Aldrich) and 2% laminin (Invitrogen) at 37°C overnight and then washed six times with PBS. At day 7, mature NSs (10–20 NSs per coverslip) were transferred onto the coverslip and incubated in 2 mL differentiation medium (90% DMEM/F12 and 10% proliferation supplement) in each well. After 5 days of incubation, the NSs became adhered to the coverslips.
Immunofluorescent staining of mature or differentiated NSs was performed as described (Golmohammadi et al. 2008). The NSs were fixed with 4% paraformaldehyde (Amresco, Solon, OH) for 30 min at RT. After one PBS wash, they were blocked in blocking solution [PBS with 0.1% triton100 (Amresco), 5% FBS, and 5% goat serum (ZSGB-Bio, Beijing, China)] for 60 min at 37°C. After that, the NSs were firstly stained with primary antibody and then with secondary antibody in blocking solution, the nuclei were stained with 10 μg/mL Hoechst (1:1000; Beyotime, Jiangsu, China). Primary antibodies: mouse-anti-nestin (1:200; 307501, R&D, Minneapolis, MN), rabbit-anti-SOX2 (1:200; L1D6A2, Cell Signal Technology (CST), Boston, MA), mouse-anti-GFAP (1:200; GA5, CST) and rabbit-anti-ßIII-tubulin (1:200; D65A4, CST); secondary antibodies: Alexa Fluor 568 donkey-anti-mouse (1:1000; Invitrogen/molecular probe) and Alexa Fluor 488 donkey-anti-rabbit (1:1000; Invitrogen/molecular probe). The coverslips were then mounted using GVA mounting medium (ZSGB-Bio).
Animals were administered with BrdU (10 mg/mL in physiological saline; Sigma-Aldrich) twice per day by intraperitoneal injection at a dose of 350 mg per kilogram body weight, 3 days before sacrifice. After ether anesthesia, animals were transcardially perfused with 50 ml normal saline and 50 mL 4% PFA. The brains were fixed in 4% PFA overnight at 4°C, then cryoprotected in 30% sucrose and embedded in embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA). Transverse sections were cut using a cryostat (10 µm) (Leica, Wetzlar, Germany). For immunofluorescence staining, sections were re-hydrated (three PBS washes and 2 mol/L HCl for 1 h at RT). After four PBS washes, sections were treated with blocking solution (PBST and 10% goat serum) for 1 h at RT, then incubated in sheep-anti-BrdU antibody (ab1893, Abcam, Cambridge, MA) solution (1:200 in blocking solution) at 4°C overnight. After three PBS washes, sections were incubated in Alexa Fluor 488 donkey-anti-sheep antibody (1:300, Invitrogen/Molecular probe) for 2 h at RT. Following three PBST washes, the nuclei were stained with Hoechst (10 µg/mL) at RT for 20 min. The sections were mounted with GVA mounting medium. The number of BrdU-positive cells was determined from five consecutive sections (Wojtowicz and Kee 2006).
Each experiment was repeated at least twice in triplicate each time, if not otherwise specified. A one-way ANOVA was used for mean comparison. A χ2 test was used for the comparison of NS size distribution p < 0.05.
Phase contrast images of alive NSs in the culture medium were taken at RT using an inverted microscope with UPlanFl 10×PH/0.3 objective lens (Olympus IX71, Japan) and a cooled EMCCD (Andor iXon DV897, UK, 512 × 512 pixels).
Fluorescent images of immunostained NSs and sections were taken using a fluorescent microscope with Plan APO 10×/0.45 or 20×/0.75 objective lens (Nikon FXA, Japan), and a cooled CCD (Olympus DP71, Japan, 1360x1024 or 4080× 3072 pixels). G-2A (EX510-560/DM575/BA590), B-2A (EX450-490/DM505/BA520), UV-2A (EX330-308/DM400/BA420) filters (Nikon) were applied for Alexa Fluor 568, Alexa Fluor 488 and Hoechst, respectively. Images were merged with Adobe Photoshop CS4.