Phenotypic differentiation of human NSCs transduced with Olig2 transcription factor at 7 weeks following transplantation into contused spinal cord. Grafted cells were detected by immunoreactivity against human mitochondrial antigen (red). (A-C) F3.Olig2 human NSCs positioned in the spared white matter (A) were colocalized with APC-CC1 (CC-1) (B). Merged cells were shown in yellow (C). Arrows indicate the same cells in the three images. (D-E) Magnified images of a grafted F3.Olig2-derived CC1 positive cell. (Inset) a low magnification merged image showing the location of the magnified cell close to the outer rim of the spared white matter. (F) A 3D reconstruction image of the double positive cells in (D, E). (G) F3 NSCs in the white matter. In contrast to F3.Olig2 cells, F3 cells were very infrequently observed in the white matter and very few of them were positive for CC1. (H) In contrast to F3.Olig2 NSCs in the spared white matter, most of F3.Olig2 cells that remained in the gray were not positive for CC1. (I-J) F3.Olig2 NSCs in the white matter expressed myelin basic protein (MBP). Arrows indicate the same cells. (K) Most of the F3.Olig2 cells in the white matter did not differentiated into astrocytes. (L) Only a small proportion of F3.Olig2 or F3 NSCs in the gray matter were positive for GFAP (arrow) (M) Many F3.Olig2 or F3 cells around the lesion cavity still expressed NSC marker nestin (arrows). Dotted lines in (L-M) indicate the margins of lesion cavities. All scale bars = 20 μm. (N) Quantification of percent NSCs that differentiated into oligodendrocytes (CC1) or astrocytes (GFAP). Error bars indicate mean ± SD. *** = p < 0.001 by unpaired T test. N = 4 animals for each group.