Adult C57BL/6J mice were culled by cervical dislocation and the spinal cords and brains collected and dissected in ice cold sterile phosphate buffered saline (PBS 1X). After carefully removing the meninges, whole spinal cords were cut into small pieces with a blade and dissociated in digestion medium (EBSS 1X, 200 mg/L Cysteine, 200 mg/L EDTA (Sigma), 2 U/ml Papain) for 45 min at 37°C.
After centrifugation, the pellet was mechanically dissociated and re-suspended in 0.9 M sucrose in EBSS, centrifuged again and subsequently resuspended in 200 μl of Complete Growth Medium (CGM, mouse NeuroCult basal medium, Stem Cell Technologies Catalog #05700 with the addition of mouse NeuroCult proliferation supplements, Stem Cell Technologies Catalog #05701) in the presence of heparin 0.002% (Sigma #H3393), basic fibroblast growth factor (bFGF) (10 ng/ml) (Peprotech #100-18B-1000), epidermal growth factor (EGF) (20 ng/ml) (Peprotech #AF-100-15-1000) and antibiotics (Pen/Strep, Invitrogen #1514012). The cell suspension was slowly added on top of 4% BSA in EBSS, and centrifuged 7 min at 200 g. The myelin-free pellet was then resuspended in CGM, plated in a 25 cm2 tissue culture flask at the density of 8000 cells/cm2, and incubated at 37°C, 5% CO2. After the first 6 passages of mechanical dissociation, cells were enzymatically dissociated once every 4–5 days with Accumax (Ebioscience 00-4666-56) for 10 min at 37°C and plated at 8000 cells/cm2.
Starting from passage 10, NSCs were grown in a glutamate free, custom-made medium (NB medium) composed of Neurobasal (Invitrogen, #12349015), bovine serum albumin 200 g/l, N2 supplements (Thermo Fisher Scientific 17502048), heparin, EGF (20 ng/ml), FGF (10 ng/ml), antibiotics (Pen/Strep, Invitrogen #1514012) and 2 mM Glutamine (35050-038). DHPG (#0805), AP5 (#0105), CNQX (#1045), MPEP (#1212) and LY 367385 (#1237) were purchased from Tocris Bioscience. As for SVZ-derived NSCs, the region containing the lateral ventricles was cut into 1 mm3 pieces and resuspended with a digestion solution containing Earle's Balanced Salt Solution (EBSS), Ethylenediaminetetraacetic acid (EDTA), Cysteine and Papain. After centrifugation, the pellet was re-suspended with complete growth medium until the formation of neurospheres.
For the differentiation protocol (Suppl. Fig. 1E-H) NSCs were collected, centrifuged, counted, resuspended in differentiation medium (CGM, mouse NeuroCult differentiation medium, Stem Cell Technologies) and plated on Matrigel-coated glass coverslips (placed inside 24 multiwell plates) with a density of 80.000 cells/cm2 in 130 of medium. Cells were then placed in the incubator (37°C, 5% CO2) for 30 min to homogenously settle onto the coverslips. Then the wells were filled with differentiation medium and cells were incubated at 37°C, 5% CO2 for 6 days. Half of the medium was changed to fresh medium after 3 days.
Gene expression analysis
Total RNA was extracted following TRIzol® reagent (Life Technologies, #15596-026) manufacturer description. 1 μg of RNA was converted into cDNA using high capacity cDNA reverse transcription kit (Applied Biosystem, #4368813). For the semi-quantitative analysis, 100 ng cDNA was amplified according to manufacturer description (Life Technologies, #10342-053) by using the primers listed in [Castiglione, #86]. Reaction conditions included an initial denaturation step (94 C/ 3 min) followed by 30–33 cycles of (94 C/45 s; 55 C/30 s; 72 C/30 s), followed by a final extension step (72 C/10 min). Samples were run using a 2% agarose gel in TBE.
For quantitative qPCR, 10 ng of cDNA was amplified using TaqMan® Fast Universal PCR Master Mix (Life Technologies, #4352042) and the following primers: Vegf (Life Technologies, Mm01281449_m1), Lif (Life Technologies, Mm00434762_g1) and Bdnf (Life Technologies, Mm00434762_g1). 96 well plates were used and the samples read with a 7500 Fast Real-Time PCR system machine (Applied Biosystems). The Ct method was used for quantification of gene expression. Expression levels were normalized to β-actin mRNA. All the experiments have been performed at least three times (from n=3 independent biological replicates).
Unless differently specified, NSCs were fixed with pre-warmed 2% PFA + 2% sucrose in PBS for 5–10 min at RT and subsequently washed 3 times with PBS and conserved at 4°C with 0.005% PBS sodium azide. Fixed cells were then incubated with blocking solution [PBS + 10% Normal Goat Serum (NGS, PAA #B11-035)] with 0.3% Triton X-100, for 1.5 h at RT. The blocking solution was then removed and the cells were incubated either O/N at 4°C or 2 h at RT with the primary antibody (see Suppl. Info.) diluted in PBS-NGS 1% with 0.3% Triton X-100. Cells were then washed twice with PBS and incubated 1 h at RT with the appropriate Alexa Fluor 488, 546 or 647 secondary antibody diluted 1:1000 in PBS-NGS 1% with 0.3% Triton X-100. Cells were washed three times in 1X PBS and incubated for 3 min with 4',6-diamidino-2-phenylindole (DAPI) at RT in the dark. Finally, cells were washed twice with PBS, once with distilled water and mounted on glass microscope slides with mounting medium (DAKO, #S3023). Slides were stored at 4 or -20°C.
ELISA protein binding assay
ELISA protein binding assays were performed according to the manufacturer's protocol (R&D DBNT00 and R&D MLF00). Medium samples were previously collected and stored at -80°C and used undiluted. Absorbance was measured at 450 nm using a microplate reader (Infinite M200 Pro-TECAN). Concentrations were extrapolated by using GraphPad Prism 4.0 software.
Ca2+ imaging analysis
NSCs were seeded at a density of 100.000 cells/cm2 on laminin coated-glass bottom culture dishes (MatTek Corporation) and primed for 3–5 days with a medium containing Neurobasal (Gibco 21103-049), N2 supplement (Thermo Fisher Scientific 17502-048), 10 ng/ml bFGF (PeproTech #100-18B-1000), 1 μg/ml laminin (Roche 11243217001), 5 μg/ml heparin (Sigma #H3393), 1.5% glucose, glutamine (Thermo Fisher Scientific 35050-038), and antibiotics (Pen/Strep, Invitrogen #151401). NSCs were loaded with 5 μM Fluo-4AM (Life Technologies, F-14217) for 30 min at 37°C and washed twice for 15 min at 37°C with Tyrode’s solution, an isotonic solution resembling the composition of the cerebrospinal fluid and containing 129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 3 mM MgCl2, 30 mM Glucose, 25 mM Hepes. The chamber was then mounted on the stage of a Leica DMI 6000B inverted live imaging microscope within a heated (37°C) and CO2 conditioned box. Images were acquired with a frequency of 2 frames per second (fps). The chamber was connected with a perfusion system to allow a continuous and regular flow of solutions when necessary. NSCs were recorded for a total of 160 s: 15 s of basal recording, 105 s of stimulation and 40 s of wash out. Cells were also imaged for the same time length during basal conditions to quantify their Ca2+ spontaneous activity at the beginning and at the end of each experimental session. Each acquired time-lapse was then analyzed with a custom-made macro using the software Fiji. Individual regions of interest (ROIs) corresponding to the soma of each imaged cell were automatically acquired. Additional ROIs were manually added into 5 cell-free areas of the imaged field to compensate for fluorescent background. The fluorescent changes over time within each ROI were then measured and the average background subtracted. The first 10 s of recording were used to calculate the average basal fluorescence (F0). Fluorescence values for each time frame were then calculated as F= ΔF/F0, where ΔF is Fi-F0, with Fi being the fluorescence value of an ROI at any given time point. Fluorescent values were then plotted over time. The number of active cells was calculated as the percentage of cells showing a ΔF/F0 > 0.4 in at least 5 consecutive frames (2.5 s).
Statistical analyses were performed using GraphPad Prism 4.0 software. One-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test correction was used for multiple group comparison and repeated measure two-way ANOVA was used to analyze differences in behavioural tests, in not differently indicated.