Figure 4.

NPCs detached from the Nichoid retain a memory of pluripotency induction. A. NPCs were grown in standard floating conditions or inside the 3D scaffold for 7 days, then replated (1x10⁴ cells/well) in standard floating conditions for 7 more days (please see schematic). Cells were counted with trypan blue exclusion method (blind observer). Data are expressed as mean of three independent experiments ± SD (**p <0.01 vs Control). B. Representative immunofluorescence images of NESTIN, BETA-TUBIII, GFAP in replated NPCs. Nuclei were stained in blue (DAPI). Scale bar 20 µm. The adjacent panels show the markers' distribution along the Z axis. The quantification of fluorescence intensity was made with ImageJ software. C. mRNA expression levels of Sox2, Oct4 and Nanog in replated NPCs. Data are expressed as mean of three independent experiments, each performed in duplicate ± SEM (n=6, *p <0.05, **p <0.01 vs Control; #p < 0.05; ##p <0.01 vs NPCs at day 0). D. Representative immunofluorescence images of NESTIN, GFAP, BETA-TUBIII, MAP2 and NG2 of NPCs grown for 7 days inside the Nichoid and then differentiated for 7 more days. Nuclei are stained in blue (DAPI) and the other markers in red. Scale bar 20 µm. The graphs report the quantification of fluorescence intensity made with ImageJ software. Pictures are representative of three different experiments. Data has been reported as mean ± SD (n=3, * p < 0.05, **p <0.01 and ***p <0.001 vs Control). Neurite length made with ImageJ was not significantly different between the two conditions. The data reported in the diagram are referred to mean ± SD.