ES cells and neural differentiation are detailed elsewhere [38 (link)]. The LC1 and other ES cell–derived NS cells were routinely generated by re-plating d 7 adherent neural differentiation cultures (typically 2–3 × 106 cells into a T75 flask) on uncoated plastic in NS-A medium (Euroclone, Milan, Italy) supplemented with modified N2 [36 (link)] and 10 ng/ml of both EGF and FGF-2 (NS expansion medium). Over 3–5 d, cells formed aggregates that, after harvesting and sedimentation to remove debris, subsequently attached to fresh plastic and outgrew NS cells. After addition of 0.5 μg/ml of puromycin to differentiating adherent cultures at d 7, 46C-NS cells were generated. Cells were re-plated 3 d later into an uncoated T75 flask in N2B27 media with 10 ng/ml of both EGF and FGF-2 (Peprotech, Rocky Hill, New Jersey, United States) in the absence of puromycin. To derive clonal lines, including NS-5, single cells were plated into 96-well microwell plates (Nalge Nunc International, Rochester, New York, United States) by limiting dilution. and the presence of one cell per well was scored 1 h after plating.
For derivation directly from foetal CNS, primary cultures were generated using standard protocols from cortex or striatum of E16.5 mouse embryos and subsequently allowed to attach on flasks treated with 0.1% gelatin. Outgrowing cells were then expanded on gelatin using NS expansion medium. Clonal derivatives of the cortical line Cor-1 were established by plating at very low density (1,000 cells per 9-cm plate) and expanding individual colonies.
For derivation from established neurospheres, derived from E14 foetal brain and maintained for 40 passages in EGF plus FGF-2, cultures were dissociated to single cells using Accutase (Sigma, St. Louis, Missouri, United States) and plated at 104 cells/ml on gelatin-coated culture flasks in NS expansion medium.
For passaging established NS cell lines, we routinely used trypsin/EDTA or PBS and split cells 1:3 to 1:5 every 2–3 d. For astrocyte differentiations, NS cells were re-plated onto 4-well plates at 1 × 105 cells/well in NS-A medium supplemented with 1% fetal calf serum or 10 ng/ml BMP4 (R&D Systems, Minneapolis, Minnesota, United States). For neuronal differentiation, 5 × 104 NS cells were plated into poly-ornithine/laminin treated wells in NS-A supplemented with FGF-2 alone. After 7 d, the media was switched to NS-A supplemented with B27 (GIBCO, San Diego, California, United States) without growth factor. Half of the medium was exchanged every 2–3 d during the differentiation. For clonal differentiation, 1,000 cells from NS-5 or Cor-1, cultures were plated in 10-cm plates pre-treated with laminin, expanded for 12 d in EGF/FGF-2, and differentiated in situ as above. For electrophysiological studies, 1.5 × 105 NS cells were plated into poly-L-ornithine-treated 35-mm dishes in NS-A medium supplemented with N2 and B27 (both at 0.5%) and FGF-2 (5 ng/ml). After 7 d, the medium was switched to the mix NS-A:Neurobasal (1:1), supplemented with B27 (GIBCO) without growth factors. To sustain neuronal maturation, after a further 7 d, the medium was switched to the mix NS-A:Neurobasal (1:3) supplemented with B27 (GIBCO) and brain derived neurotrophic factor (20 ng/ml) and nerve growth factor (R&D Systems; 50 ng/ml). Throughout neuronal differentiation, half of the medium was replaced every 2–3 d. Further details of NS cell derivation, propagation, and differentiation are provided inProtocol S1 .
For derivation directly from foetal CNS, primary cultures were generated using standard protocols from cortex or striatum of E16.5 mouse embryos and subsequently allowed to attach on flasks treated with 0.1% gelatin. Outgrowing cells were then expanded on gelatin using NS expansion medium. Clonal derivatives of the cortical line Cor-1 were established by plating at very low density (1,000 cells per 9-cm plate) and expanding individual colonies.
For derivation from established neurospheres, derived from E14 foetal brain and maintained for 40 passages in EGF plus FGF-2, cultures were dissociated to single cells using Accutase (Sigma, St. Louis, Missouri, United States) and plated at 104 cells/ml on gelatin-coated culture flasks in NS expansion medium.
For passaging established NS cell lines, we routinely used trypsin/EDTA or PBS and split cells 1:3 to 1:5 every 2–3 d. For astrocyte differentiations, NS cells were re-plated onto 4-well plates at 1 × 105 cells/well in NS-A medium supplemented with 1% fetal calf serum or 10 ng/ml BMP4 (R&D Systems, Minneapolis, Minnesota, United States). For neuronal differentiation, 5 × 104 NS cells were plated into poly-ornithine/laminin treated wells in NS-A supplemented with FGF-2 alone. After 7 d, the media was switched to NS-A supplemented with B27 (GIBCO, San Diego, California, United States) without growth factor. Half of the medium was exchanged every 2–3 d during the differentiation. For clonal differentiation, 1,000 cells from NS-5 or Cor-1, cultures were plated in 10-cm plates pre-treated with laminin, expanded for 12 d in EGF/FGF-2, and differentiated in situ as above. For electrophysiological studies, 1.5 × 105 NS cells were plated into poly-L-ornithine-treated 35-mm dishes in NS-A medium supplemented with N2 and B27 (both at 0.5%) and FGF-2 (5 ng/ml). After 7 d, the medium was switched to the mix NS-A:Neurobasal (1:1), supplemented with B27 (GIBCO) without growth factors. To sustain neuronal maturation, after a further 7 d, the medium was switched to the mix NS-A:Neurobasal (1:3) supplemented with B27 (GIBCO) and brain derived neurotrophic factor (20 ng/ml) and nerve growth factor (R&D Systems; 50 ng/ml). Throughout neuronal differentiation, half of the medium was replaced every 2–3 d. Further details of NS cell derivation, propagation, and differentiation are provided in
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