Fig. 2

Chi3l3 directly promotes oligodendrogenesis in vitro. Representative confocal image (above) and quantification (below) of neural stem cells (NSCs) cultured in the presence of PBS (control) or Chi3l3 (100 ng/ml) for 3 days a–d, 5 days e–h, or primary OPCs cultured in the presence of PBS (control) or Chi3l3 (500 ng/ml) for 7 days i. Cells were treated with the nuclear stain TO-PRO-3 (blue) and immunostained for early progenitor markers NG2 (a; oligodendrocyte precursor cells, green), GFAP (b; astrocytes, green), Dcx (c; neuroblasts, green), late progenitor markers O4 (e; oligodendrocytes, green), GFAP (f; astrocytes, green) and microtubule-associated protein 2 (g; Map2, neurons, green), the neural stem cell marker Sox2 (d, h; green) and the myelin protein MBP (i, oligodendrocytes, green). Exposure of differentiating NSCs to Chi3l3 led to significant increase in oligodendrocyte precursor cells and oligodendrocytes, significant decrease in astrocytes, neuroblasts, and neurons and a significant increase in Sox2⁺ neural stem cells. Scale bar, 50 μm. Inserts show representative cells. Scale bar, 20 μm. j–n Gene expression of Cspg4 (NG2; j), Gfap k, and Map2 (l; 3 days) and Ccnd1 and Ccnd2 (m, n; 24 h) mRNA in PBS (control) or Chi3l3-treated differentiating NSCs. Values were normalized against Gapdh (AU, arbitrary unit; n.s., not significant;). Number o and size p of neurospheres from NSCs exposed to Chi3l3 or PBS (control). (n.s., not significant; two-tailed Student’s t test; data are representative of three independent experiments with n = 5 replicates a, c, d, i, n = 9 (Control) and 10 (Chi3l3) replicates b, n = 12 (Control) and 9 (Chi3l3) replicates e, n = 12 replicates f, g, n = 3 replicates h, j, l, m, n), n = 3 (Control) and 2 (Chi3l3) replicates k, n = 4 replicates o, n = 8 (control) and four (Chi3l3) replicates p). mean ± s.e.m. *p < 0.05; **p < 0.01; ***p < 0.005