Neuronal differentiation of Df stem cells. Protocol of neuronal differentiation. DF stem cells were seeded in 6 well plates at cell density of 20 × 105 cells/well. When cells reached confluence a two steps protocol was applied: cells were cultivated for 48 h in presence of neuronal differentiation medium 1 consisting of DMEM high glucose/F12-HAM (1:1 ratio), 10 % fetal bovine serum (FBS), 1 % antibiotics,2 mM glutamine, 1 % non-essential aminoacids (NEA), supplemented with 10 ng/ml Epidermal Growth Factor (EGF), 10 ng/ml basic Fibroblast Growth Factor (bFGF), 2 % B27 and 1 % N2 Supplement. Afterwards cells were exposed to the differentiation medium 2 for 3 weeks: DMEM high glucose/F12-HAM, 10 % FBS, 1 % antibiotics, 2 mM glutamine, 1 % NEA, 1 % N2 Supplement, 2 % B27 Supplement, 3 μM all-trans retinoic acid, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX) (all reagents were purchased from Sigma Aldrich). At the end of 3 weeks of DF stem cultivation with neuronal differentiation medium, cells were fixed and immunocytochemical stained for neurofilaments and CD 133 expression. As shown in Figure S1, the stained cells expressed positivity only for neurofilaments. Figure S1: Fluorescence image of DF stem cells induced to differentiate into neuronal cells. Cells were stained with anti neurofilaments antibody conjugated with FITC green), anti CD 133 conjugated with Texas red (red) and nuclei were counterstained with DAPI (magnification ×100). (PNG 451 kb
