FIG. 2.

Morphology and growth curve of proliferating NPCs. Phase contrast images of NPCs in normal proliferation medium (control) (A, E, I), treated with 50% MSC-CM (B, F, J), 100% MSC-CM (C, G, K), or MSC-CM replenished with growth factors (MSC-CM+GF) (D, H, L). Proliferating NPCs were incubated for 7 days (A–D), 14 days (E–H), and 21 days (I–L). Note that under control conditions proliferating NPCs grew as floating aggregates, but when the cells were treated with MSC-CM some NPCs attached to the surface of the flask and grew adherent. Scale bar=100 μm. The cell number was determined by Trypan blue exclusion after 7, 14, and 21 days. Growth curves for the different conditions were determined (M). The metabolic activity of the cells (N) and the number of neurospheres (O) and the mean neurosphere diameter (P) were analyzed on day 2, 4, and 7 during the second week of incubation with the respective media. Note that there is no significant effect of MSC-CM on the NPCs proliferation rate. All experiments were performed at least in triplicate in 3 independent experiments. Data are shown as mean±SD. For statistical analysis 2-way ANOVA-Bonferroni post hoc was performed. SD, standard deviation. ANOVA, analysis of variance; GF, growth factor.