Purmorphamine

For the in vitro differentiation to CNS neurons, PSCs were detached using Accutase (Innovative Cell Technologies, AT104) for 20 min. at 37°C, washed twice with 1X PBS and plated onto matrigel (BD, 354234)-coated 6-wells in HES medium supplemented with 10 μM Y-27632 dihydrochloride (Tocris, 1254). When 100% confluency was reached the cells were fed daily with KSR- or N2-medium supplemented with LDN193189 (Stemgent, 04-0074) and SB431542 (Tocris, 1614)¹⁸ . On day 11 they were Accutased, washed twice and replated onto Poly-L Ornithine hydrobromide (Sigma, P3655)/Laminin-1 (R&D Systems, 3400-010-01)/Fibronectin (BD Biosciences, 356008)-coated plates in droplets of approximately 100–150,000 cells per 10 μl as described in Zeltner et al.¹⁸ , in N2 medium supplemented with 0.02 μg/ml BDNF (R&D Systems, 248-BD), 0.2 mM Ascorbic Acid (Sigma, A4034) and 0.1 μM Purmorphamine (Stemgent, 04-0009). On day 13 the cells were fed with N2 medium/AA/BDNF/Purmorphamine, on day 15, 17 and 19 they were fed with N2 medium/AA/BDNF/ 0.01 mM DAPT (R&D, 2634/50) and on day 20 they were fixed with 4% PFA (Affymetrix, 19943) and immuno-stained with Tbr1 (rabbit, Millipore, AB10554) and Tuj1 (mIgG2a, Covance, MMS-435P-250).

On day 12, cells were dissociated using Accutase and replated in high-density conditions (300,000 cells per cm2) on dishes precoated with polyornithine (PO; 15 μg/ml), laminin (Lam; 1 μg/ml) and fibronectin (FN; 2 μg/ml) in N2 medium supplemented with brain-derived neurotrophic factor (BDNF; 20 ng/ml, R&D), ascorbic acid (AA; 0.2 mM, Sigma-Aldrich), Purmorphamine (1 uM, Stemgent) and fibroblast growth factor 8 (FGF8; 100 mg/ml, R&D). They are patterned at the P1 stage for 2 weeks. After two weeks of patterning, cells were passaged by mechanical picking of the CNS clusters and re-plated on PO/Lam/FN coated dishes (P2). Starting with P3, neural precursor cells (NPCs) were maintained in N2/B27 medium supplemented with Purmorphamine and FGF8 until day 40 when they were further exposed to glial media containing platelet-derived growth factor (PDGF; 20 ng/ml, R&D), insulin-like growth factor 1 (IGF-1; 20 ng/ml, R&D), triiodothyronine (T3; 20 ng/ml, Sigma-Aldrich) and dibutyryl cAMP (0.1 mM, Sigma-Aldrich). Medium was changed every 2 days, whereas the cultures were passaged every 2 weeks.