Foetal mouse neural stem cells give rise to ependymal cells in vitro

NSCs are responsible for the generation of CNS cell types derived from the neural tube. Published data resulting from experiments studying the differentiation of NSCs in vitro or in vivo have confirmed their spontaneous tripotency, i.e. their ability to generate cells of the neuronal, astroglial and oligodendroglial lineages. The relationship between NSCs generated in vitro and ependymal cells has not yet been studied. To confirm that ependymal cells can also be produced by NSCs, we utilized the neurosphere assay, which permits isolation and cultivation of NSCs. Cells from the forebrain of E14-15 Balb/c foetuses were grown in DMEM/F12-N2 medium supplemented with EGF and FGF-2 to form multicellular neurospheres. After 3 to 8 passages, neurospheres were plated on surfaces coated with poly-L-lysine, polyornithine and/or laminin in dishes containing the same medium where cytokines were replaced with serum. Under these conditions, neurosphere cells spread over the surface forming a cellular layer consisting of beta-III tubulin+ neuronal, GFAP+ astroglial and O4+ oligodendroglial cells. When these cells were cultivated for prolonged periods, they formed islands of epitheloid cells. Following 2 to 3 weeks in vitro, ependymal cells with beating cilia appeared among these cells. Ciliated ependymal cells were observed in small clusters or as single cells scattered in certain areas. Confocal microscopy confirmed the presence of alpha-tubulin-immunoreactive cilia arranged in tufts located on the apical surface of epitheloid cells. Our data indicate that ependymal cells are spontaneously derived from NSCs.