Figure 4.

CD271⁺/GFAPδ⁺ cells form neurospheres and differentiate into astrocytes, neurons, and oligodendrocytes. (A): CD271⁺/GFAPδ⁺ cells were cultured in a neurosphere assay, and pictures were taken at days 1, 6, and 12 after isolation. (B): At day 15 after isolation, neurospheres were collected and allowed to differentiate for 14 days. CD271⁺/GFAPδ⁺ cells were able to differentiate into GFAP-positive astrocytes (green) and βIII-tubulin-positive neurons (red) (C) and GalC-positive oligodendrocytes (D), as shown with double-immunofluorescent staining. (E): Some cells are positive for both GFAP and βIII-tubulin. (F): Primary neurospheres were dissociated and plated as single cells per well. Small new spheres were formed after 2 weeks. (G): A single neurosphere differentiated into GFAP-positive astrocytes (green) and βIII-tubulin-positive neurons (red). (H): New neurospheres were formed after dissociation of secondary single neurospheres. (I): These cells were plated onto laminin-coated wells and allowed to adhere. Then cells were immunofluorescently stained for GFAPδ. Scale bars represent 50 μm (B–H) or 100 μm (I). Abbreviations: GalC, galactoceramide; GFAP, glial fibrillary acidic protein.