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Materials and methods
Astrocyte and tuberous sclerosis complex cell cultures
Primary fetal astrocyte-enriched cell cultures were obtained from human fetal brain tissue (cortex, 14-19 gestational weeks) obtained from medically induced abortions. All material has been collected from donors from whom a written informed consent for the use of the material for research purposes had been obtained by the Bloemenhove clinic. Tissue was obtained in accordance with the Declaration of Helsinki and the Academic Medical Center (AMC) Research Code provided by the Medical Ethics Committee of the AMC. Cell isolation was performed as described previously (17 see supplementary material). TSC cell cultures were derived from surgical brain specimens obtained from 2 patients (age at surgery: 2.5 and 2 years; gender: male; mutation: TSC2) undergoing epilepsy surgery at the Wilhelmina Children’s Hospital of the University Medical Center Utrecht (UMCU, Utrecht, The Netherlands). TSC cultures were established in the same manner as fetal cultures.
Neural stem cell cultures
Neural stem cells (NSCs) were obtained from fetal brain (14-16 gestational weeks). Tissue was enzymatically digested by incubating at 37˚C for 30 minutes with 0.3% tryp- sin (Sigma-Aldrich; St. Louis, MO, USA). The reaction was stopped by the addition of fetal calf serum (FCS). Cells were washed and taken up in complete deficient medium (dDMEM; DMEM without phenol with 10% FCS and 1% P/S) and triturated through a 70 μm mesh filter. NSCs were selected by a five-step discontinuous density gradient separa- tion. 100% standard isotonic Percoll (SIP, 9 parts Percoll, GE Healthcare, Auckland, New Zealand, with 1 part 10x PBS pH 4.6) was diluted to 50, 40, 30, 20 and 10% with dDMEM and these dilutions were layered starting with the highest concentration at the bottom and the cells on top. After centrifugation, the cells at the 30/40% SIP interphase were collected and grown at 37˚C, 5% CO2 in proliferative medium (DMEM/HAM F10 (1:1) supplemented with 2% B27 (50x), 20 ng/ml EGF, 20 ng/ml bFGF (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% P/S). Medium with EGF and bFGF was refreshed every 2-3 days. For transfection and differentiation experiments, NSCs were re-plated on laminin (10 μg/ml, Sigma-Aldrich). For differentiation of NSCs, medium was replaced by DMEM/HAM F10 (1:1) with 1% P/S and 5% FCS. To quantify differentiation, βIII-tubulin positive cells were counted , and the number of 4’,6-diamidino-2-phenylindole (DAPI) positive nuclei was determined with particle analysis (both with ImageJ 1.44p, National Institutes of Health, Bethesda, MD, USA).
Transfection and stimulation of cell cultures
Cells were transfected with mimic pre-miRNA for miR146a or miR147b (mirVana miRNA mimics, Applied Biosystems, Carlsbad, CA, USA) for 24 hours as described previously 17. Astrocyte cultures were stimulated with human recombinant (r)IL-1 β (10 ng/ml; Peprotech, Rocky Hill, NJ, USA) or lipopolysaccharide (LPS; 100 ng/ml; Sigma, St. Louis, MO, USA) for 24 hours. Viability of human cell cultures was not influenced by the treat- ments (as shown previously; 21). To examine the effect of the IκB kinase-2 (IKK-2) inhib- itor TPCA-1 in astrocyte cultures, treatment with 1 or 5 μM TPCA-1 (Selleck Chemicals, Munich, Germany) in DMSO (0.05% final DMSO concentration) was started 1 hour before


























































































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