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ES cells [line J1 (21), passage number ≤ 17] were grown on γ-irradiated embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, 0.1 mM 2-mercaptoethanol, nucleosides, nonessential amino acids, and human recombinant leukemia inhibitory factor (LIF, Life Technologies; 1000 units/ml). Cells were passaged once onto gelatin-coated dishes and then aggregated to form embryoid bodies in the absence of LIF. We plated 4-day-old embryoid bodies in tissue culture dishes and propagated them for 5 days in ITSFn medium [DMEM/F12 supplemented with 5 μg/ml insulin, 50 μg/ml transferrin, 30 nM selenium chloride, and 5 μg/ml fibronectin (8)]. Cells were then trypsinized, plated in polyomithine-coated dishes (15 μg/ml), and propagated in DMEM/F12 supplemented with insulin (25 μg/ml), transferrin (50 μg/ml), progesterone (20 nM), putrescine (100 μM), selenium chloride (30 nM) plus FGF2 (10 ng/ml), and laminin (1 μg/ml). After 5 days, cells were harvested by scraping in calcium- and magnesium-free Hanks' buffered salt solution (CMF-HBSS). triturated to a single-cell suspension, replated at a 1:5 ratio, and grown to subconfluency in the presence of FGF2 (10 ng/ml) and ECF (20 ng/ml). Cells were then passaged at a 1:5 ratio and again grown to subconfluency in the presence of FGF2 (10 ng/ml) and PDGF-AA (10 ng/ml). Human recombinant FGF2, EGF, and PDGF-AA (R&D Systems, Minneapolis, MN) were added daily and the medium was replaced every 2 days. In vitro differentiation was induced by growth factor withdrawal.
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0345151192
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note
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Astrocytic and oligodendroglial differentiation were assessed by immunofluorescence analysis using antibodies to A2B5 (Boehringer; 1:200), O4 (Boehringer; 1:5) and GFAP (ICN; 1:100). Similar results were obtained using the ES cell line CJ7 (22) (passage number ≤ 18). Four days after growth factor withdrawal, 31.4 ± 3.6% and 35.0 ± 7.4% of the cells were immunoreactive to the O4 and GFAP antibodies, respectively (n = 5).
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0344720895
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Cells growing in medium containing FGF2 and PDGF were harvested and triturated to a single-cell suspension. Spinal cord transplants were performed as described (23). Following laminectomy at the thoracolumbar transition, 100,000 cells in a volume of 1 μl CMF-HBSS were injected into the dorsal column of 7-day-old md rats at one or several levels. The recipient animals received dally i.p. injections of cydosporin (10 mg/kg body weight). All procedures were done in accordance with institutional guidelines.
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Transplant recipients were perfusion-fixed with 4% paraformaldehyde at 3 weeks of age. Brains and spinal cords were processed for vibratome sectioning. Selected spinal cord transplant recipients were processed for semi- and ultrathin sectioning by routine procedures. Primary antibodies used for immunohistochemistry were anti-PLP (1:500), anti-MBP (Boehringer; 1:200), anti-CNP (Sigma; 1:200), and anti-GFAP (ICN; 1:100). Antigens were visualized using appropriate peroxidase-conjugated secondary antibodies. Identification of donor cells by DNA in situ hybridization with a probe to mouse satellite DNA was done as described (16).
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note
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We thank I. Griffiths for the PLP antibody and R. Buschwald for excellent technical assistance. This work was supported by a Junior Research Award from the state of Nordrhein-Westfalen (O.B.), the BONFOR program (O.B.), and NIH grant NS33710 (I.D.D.).
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