Tripotential differentiation of NS cells in vitro and generation of myelinating oligodendrocytes in vivo.

<p>(A–E) Quantitative marker expression and representative immunofluorescence images. The specific culture conditions used to differentiate NS cells resulted in the generation of oligodendrocytes (∼20%) positive for O4 (B), Rip (D) and PLP (E), GFAP-expressing astrocytes (∼40%; C–D) and neurons positive for ß-III tubulin/TUJ1 (∼10%; C). (F–H) NS cells cultured in N2 medium and proliferated for 4 days in the presence of FGF2, PDGF and forskolin were transplanted into the brain of 2- to 3-day-old myelin-deficient rats. Two weeks after transplantation, the engrafted cells had formed PLP-positive myelin internodes. Shown are representative pictures from septum (F) and corpus callosum (G–H). Scale bars B–D, 100 µm; F–H, 20 µm.</p

Protocol for the generation of oligodendrocytes from NS cells.

<p>NS cells propagated in NS-A medium plus N2 in the presence of EGF and FGF2 (A) were cultured in DMEM/F12 plus N2 in the presence of FGF2, PDGF and forskolin for 4 days on polyornithine/laminin coated plastic (B) before they were induced to differentiate by growth factor withdrawal in the presence of 3,3,5-tri-iodothyronine hormone (T3) and ascorbic acid (AA) (C,D). After four days, immunostaining for the O4 antigen revealed differentiation into oligodendrocytes (C). The differentiated cultures also contained GFAP-positive astrocytes and ß-III tubulin/TUJ1-positive neurons (C,D), demonstrating the tripotential differentiation capacity of these cells.</p