Dibutyryl camp

PBS, HBSS, DMEM/F12 (Thermo Fisher Scientific, Inc.), UW solution (Viaspan^®; Bristol-Myers Squibb, Co.), ET-Kyoto solution (Otsuka Pharmaceutical Factory, Inc.), and Optisol GS™ (Bausch & Lomb, Rochester, NY) were prepared as candidates for a basal preservation medium. The following additives were also prepared: dextran 40 (0.2, 2, 20 g/L; Wako), chondroitin sulfate A (0.01, 0.1, 1%; Sigma-Aldrich, St. Louis, MO, USA), N-acetylcysteine (0.1, 1, 10 mM; Wako), allopurinol (0.1, 1, 10 mM; Tokyo Chemical Industry Co., Ltd), glutathione (0.3, 3, 30 mM; Sigma-Aldrich), adenosine (0.05, 0.5, 5 mM; Sigma-Aldrich), hyaluronic acid (0.01, 0.1, 0.5%; Calbiochem), dibutyryl cAMP (0.2, 2, 20 mM; Enzo Life Sciences), trehalose (12, 120, 1200 mM; Hayashibara), tert-butylhydroquinone (tBHQ; 0.1, 1, 10 μM; Sigma-Aldrich), oltipraz (1, 10, 100 μM; Sigma-Aldrich), and ebselen (1, 10, 100 μM; Sigma). Each of these additives was added to HBSS individually.

At passages 2 and 5, cells were dissociated to single cells and plated for differentiation as described above. We chose this approach in contrast to direct differentiation of NSPHs for a more standardized differentiation and better quantification by microscopy. When confluent, differentiation was initiated by withdrawal of FGF-2, EGF and heparin. To enhance neuronal differentiation, we added BDNF (brain-derived neurotrophic factor, 20 ng/ml), GDNF (glial cell-derived neurotrophic factor, 10 ng/ml), IGF1 (insulin-like growth factor 1, 10 ng/ml) (all from Peprotech, Hamburg, Germany), dibutyryl-cAMP (500 μM; EnzoLife Sciences, Lörrach, Germany) and the notch inhibitor DAPT (10 μM; Tocris, Bristol UK) for 30 days.