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  • DTI and Ki67 Immunohistological Screening of Human Tissue: Twenty-four samples of adult human brain white matter of normal appearance were obtained from planned resections during anterior temporal lobectomy for the treatment of intractable epilepsy. The procedures were conducted at Hospital La Fe (Valencia, Spain) and were performed with informed patient consent that provided their written consent to participate and in accordance with the medical and science ethics board (Comité Ético de Investigacón Biomédica del hospital Universitario La FE (CEIB)) that specifically approved the present study. In order to ensure the absence of abnormalities in the tissue samples used in the present study, we first analysed the samples with two different techniques: pre-surgical diffusion tensor imaging (DTI) and automatised Ki67 immunostaining. DTI was used to infer ultrastructural changes in brain white matter tissue according to the diffusion of water molecules. The diffusion-weighted images from all the patients included in this study were acquired using a single-shot echo-planar imaging pulse sequence with the following parameters: TR/TE of 10.100/102, acquisition matrix of 128×128, FOV of 250 mm, in-plane resolution of 2×2 mm and contiguous 2-mm slice thickness, 70 contiguous axial slices, and a voxel volume of 1.95×1.95×2 mm. The images were acquired with diffusion weighting in 30 non-collinear directions, all with a b value (diffusion weighting factor) of 1000 s/mm2. In addition, an image with no diffusion weighting (b value of 0 s/mm2) was acquired as a reference. The DTI images were analysed using DTV.II SR toolbox software (Image Computing and Analysis Laboratory [UTRAD/ICAL] Department of Radiology, The University of Tokyo Hospital, extension of Volume 1, package [http://www.volume-one.org/]) with the following parameters to determine the degree of sensitivity for generating DTI tracts: Fractional Anisotropy (FA)  = 0.05, steps  = 160; Apparent Diffusion Coefficient (ADC) (x1K): any; So <80; Angle: any. The Ki67 immunohistochemistry was performed on a benchmark ULTRA using clone 30-9 (Ventana Medical Systems, Tucson, AZ), and interpreted with an iScan Coreo and Virtuoso algorithm (Ventana Medical Systems). A histopathological analysis of the tissue samples was performed to validate their use in the rest of the study. The architectural disorganisation of the cortex and neuronal heterotopia were studied using NeuN immunostaining. Microglia activation was examined by Iba-1 marking. The experiments were performed using the protocols described later. Cell Isolation Protocol and Culture: All samples were processed within 12 hours of extraction following a previously described protocol [24]. Briefly, brain white matter was macroscopically separated from temporal grey matter (Figure S1), minced, and washed in HBSS (Hanks balanced salt solution) w/o Ca2+/Mg2+. Enzymatic digestion was sequentially performed with Solution I (Papain [14 U/mL, Sigma] and DNase I [10 U/mL, Sigma] in PIPES solution) for 90 minutes at 37°C and Solution II (Papain [7 U/mL] and DNase I [15 U/mL] in PIPES: proliferative media [1∶1]) for 30 minutes at 37°C. The cells were then dissociated using diameter-decreasing polished Pasteur pipettes and filtered through a 70-µm mesh. Foetal bovine serum (10%, Invitrogen) was added to stop enzymatic digestion. In 10 of the processed samples, the tissue disaggregates were resuspended in 0.9 M sucrose in 0.5x HBSS w/o Ca2+/Mg2+ and centrifuged at 750 g for 10 minutes [25]. The dissociated cells were resuspended in defined proliferative media and were maintained using standard procedures. Four viable cell cultures were obtained without sucrose gradient separation and 10 cell lines from the procedure with sucrose gradient. The dissociated cells were resuspended in defined proliferative media composed of DMEM-F12, non-essential amino acids (100 µm), HEPES (2 mM), D-glucose (30 mM), bovine serum albumin (0.001%), sodium pyruvate (1 mM), l-glutamine (2 mM), N2 supplement (1X), penicillin-streptomycin-fungizone (1X) (all from Gibco-Invitrogen); and hydrocortisone (300 ng/mL) and triiodothyronine (30 ng/mL) from Sigma. The medium was supplemented with basic fibroblast growth factor (bFGF) (10 ng/mL, Sigma) and epidermal growth factor (EGF) (10 ng/mL, Sigma), and was replaced every two days. When the cells growing in the monolayer adherent culture reached a confluence near 90%, a subculture was performed using Triple Express (Invitrogen) for 5 minutes at 37°C. The cells were reseeded at a density of 2000 cells/cm2 in 25 cm2 culture flasks. Using this procedure, all the cell lines could be expanded for at least seven passages. The sphere-like clusters obtained using the protocol with the sucrose centrifugation were passaged before becoming necrotic by mechanical disaggregation with polished Pasteur pipettes, and planted at 3000 cells/cm2 on 24-well plates. All the cell lines could give rise to tertiary neurospheres. Additionally, adherent culture colonies derived from neurosphere cultures were subcultured separately according to monolayer culture protocol, and were able to reach a minimum of five passages. Three adherent culture cell lines produced by the spheres were seeded on ultra-low attachment at a density of 3000 cells/cm2, with or without 100-µM β-mercaptoethanol (Sigma) to test if they were able to regenerate floating cultures [26]. Viability was studied using Calcein/I Propidium iodide. After two weeks, the cells were seeded on Matrigel-coated coverslips, cultured for 24 hours, and fixed for SOX2 immunostaining (Figure 1A displays a schematic representation of the different cell cultures). Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0099253.g001 Proliferative cells can be isolated from adult human white matter.A. Diagram depicting the different procedures followed in the present work for the isolation of proliferative cells from human white matter. B. The cell isolation protocol without (w/o) the sucrose centrifugation step gives rise to a monolayer culture, which could be expanded more than seven passages. However, only 28.57% of samples generated viable cultures with this procedure (B). Therefore, we added a final sucrose centrifugation, which allowed the obtaining of sphere-like colonies lightly adhered to the culture plate from all samples processed (D). These cells generated an adherent culture that could be expanded independently for more than five passages (E). When the initial spheres were passaged and reseeded at a cell density of 3000 cells/cm2 or higher, new spheres were obtained (C), which, in turn, generated a new monolayer culture. These spheres could be passaged up to tertiary spheres, similarly to other proliferative cells isolated from adult human white matter. The scale bars represent 50 µm. Spontaneous differentiation was assayed on small spheres formed two days after disaggregation, corresponding to four samples in triplicate. The spheres were seeded on matrigel-coated coverslips and maintained in growth media for 14 days prior to fixation. A protocol directed to induce final oligodendroglial differentiation was tested in four samples in triplicate as previously described. The spheres were disaggregated into a single-cell suspension and seeded on a Matrigel-coated coverslip. The cells were maintained in an oligodendrocyte proliferative medium until confluence, composed of DMEM-F12, non-essential amino acids (100 µm), HEPES (2 mM), D-glucose (30 mM), bovine serum albumin (0.001%), sodium pyruvate (1 mM), l-glutamine (2 mM), N2 supplement (1X), and penicillin-streptomycin-fungizone (1X), all of which were acquired from Gibco-Invitrogen. The medium also included progesterone (300 ng/mL) and sodium selenite (30 ng/mL), which were acquired from Sigma. The medium was supplemented with bFGF (20 ng/mL, Sigma), platelet-derived growth factor alpha (PDGFa, 20 ng/mL, Sigma), and Neurotrophin-3 (2 ng/mL, Sigma). The medium was replaced every two days. When the cells reached confluence, the medium was supplemented with foetal bovine serum (2%, Gibco) and triiodothyronine (30 ng/mL, Sigma), and the cells were maintained one more week prior to fixation. Twenty-four hours after cell isolation with the protocol, including the centrifugation in the sucrose solution, three cell samples were lightly disaggregated with polished Pasteur pipettes and washed once with 0.1 M PBS. The cell suspension was then incubated in A2B5 hybridoma supernatant (ATCC) for 30 minutes at 4°C, washed with 0.1 M PBS, 0.5% bovine serum albumin three times, and finally incubated in a secondary antibody (Alexa Fluor 488 goat anti-mouse, 1∶500 in 0.1 M PBS [Invitrogen]) and propidium iodide. After a final wash, the samples were analysed using a Cytomics FC 500 (Beckman Coulter) flow cytometer. The total RNA was extracted from three cell lines of each culture type in passage 2 (spheres) or passage 3 (adherent cultures) using RNeasy, followed by DNase I treatment, both according to the manufacturer’s specifications (Qiagen). The cDNA was synthesised using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The molecular analyses were performed by PCR amplification, using primers designed with Primer 3 software (Table S1), followed by electrophoresis in 1.8% agarose gel. SYBR Green-based QRT-PCR (Applied Biosystems) was run in a LightCycler 480 Instrument (Roche). The amount of SOX2 cDNA was normalised to the quantity of three housekeeping gene (β-actin, β-2-microglobulin, and GAPDH) transcripts. The cells were plated for 24 hours on Matrigel-treated coverslips, fixed with 4% paraformaldehyde in 0.1 M PB (Panreac) for 20 minutes, and washed with Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen) w/o Ca2+/Mg2+. The cells were blocked with 10% donkey serum (Jackson ImmunoResearch) and 0.1% Triton X-100 (Sigma) in 0.1 M DPBS for 45 minutes. Goat anti-human SOX2 antibody (1∶50, Chemicon), mouse anti-GFAP (1∶500, Dako), mouse anti-MAP2 (1∶200, Sigma), rat anti-MBP (1∶200, Sigma), and/or mouse anti-human Ki67 antibodies (Dako, 1∶250) were applied for 1 hour at 25°C in a blocking solution. For Ki67 immunostaining (rabbit anti-human Ki67, 1∶250, Dako), antigen retrieval was previously performed by exposing coverslips to boiling 10 mM sodium citrate, 0.05% Tween 20, pH 6.0 for 15 minutes. A2B5 and O4 immunostaining was performed using live cells. The cells were washed, blocked with 5% goat serum in 0.1 M DPBS for 30 minutes at 4°C and incubated in primary antibody (A2B5 clone 105 hybridoma supernatant [ATCC, 1∶1]; anti O4 [Millipore, 1∶50]) for 45 minutes at 4°C. After washing, the cells were fixed with 4% PFA for 10 minutes and washed with 0.1 M DPBS. Finally, for all cases, after washing, secondary antibodies were applied for 1 hour in DPBS (Texas Red donkey anti-goat antibody, 1∶150; DyLight 488 Donkey anti-mouse, 1∶400 [both from Jackson InmunoResearch]; Alexa Fluor 488 goat anti-mouse IgM, 1∶500; Alexa Fluor 555 donkey anti-goat, 1∶500; Alexa Fluor 488 goat anti-rat, 1∶500; Alexa Fluor 488 donkey anti-rabbit, 1∶500; Alexa Fluor 647 donkey anti-mouse, 1∶500 [all purchased from Invitrogen]). The cells were washed, counterstained with DAPI, and mounted with FluorSave (Molecular Probes, Invitrogen). The same procedure was performed for U373 cells and/or glioblastoma multiforme cells as a positive control. The images were collected with a Leica TCS SP2 AOBS (Leica Microsystems) inverted laser scanning confocal microscope. All the confocal images were obtained under identical scan settings. Eight-bit, 1024 × 1024-pixel images were collected for each preparation. The best focus was based on the highest pixel intensity. Imaging conditions were identical for all the images. The images were identically processed using MetaMorph Software (Molecular Devices). For the cell recount, at least 10 different images (including at least five spheres, depending on their size) from three separate experiments were counted. Sections from four temporal lobe samples were fixed with 4% PFA for 36 hours. After washing, the sections were cryoprotected in a 30% sucrose solution overnight and cryosectioned at 14 µm. The sections were washed with 0.1 M PBS, and antigen retrieval was performed by immersing the sample in 10 mM sodium citrate, 0.05% Tween 20, pH 6.0 at 95°C–100°C for 1 hour. After permeabilisation treatment with 0.2% Triton X-100 in PBS for 45 minutes, autofluorescence was partially blocked using Sudan black staining. The sections were incubated overnight at 4°C with goat anti-human SOX2 (1∶25 in PBS; Chemicon), alone or in combination with mouse anti-Ki67 (1∶250, Dako), mouse anti-GFAP (1∶200, Abcam), mouse anti-NeuN (1∶800), rabbit anti-Iba-1 (1∶200, Wako), or mouse anti-CNPase (1∶200, Sigma). The samples were then washed and labelled with appropriate secondary antibodies (Texas Red-conjugated donkey anti-goat antibody (1∶150) and DyLight-conjugated Donkey anti-mouse antibody (1∶400) [both from Jackson ImmunoResearch]; Alexa Fluor 488-conjugated donkey anti-rabbit antibody [1∶500, Molecular Probes]) for 1 hour at room temperature. After washing, the nuclei were counterstained with DAPI and sections were mounted with FluorSave (Molecular Probes, Invitrogen). Surgical samples from human glioblastomas, which express high levels of SOX2, were used as a positive control (Figure S6). The images were collected with an Olympus FW-100 inverted laser scanning confocal microscope. All the confocal images were obtained under identical scan settings. Eight-bit, 1024 × 1024-pixel images were collected for each preparation. The best focus was based on the highest pixel intensity. Imaging conditions were identical for all the images. The images were equally processed using FluorView FV1000 (Olympus). The data presented in this article represent mean ± standard deviation and were calculated using Statistical Package for the Social Sciences (SPSS, IBM). The FA and ADC means from the epileptic and contralateral side were compared using Student’s t-test for paired samples (α  = 0.01).
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