Compound e

siHBs were harvested using cell dissociation buffer (0.1 mg/mL EDTA, 0.5 mg/mL BSA) and seeded onto collagen I-coated plates in RPMI/B27, 10% Fetal Bovine Serum (FBS, Gibco, Waltham, MA, USA), 1% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA), and 50 ng/mL HGF for four hours to enable cell attachment. The medium was then replaced by RPMI/ITS with 50 ng/mL HGF, 3 nM CHIR-99021 (Stemcell Technologies, Vancouver, WA, Canada) and 0.1 ng/mL TGF-β1 (Peprotech, Cranbury, NJ, USA), for 24 h. The medium was changed every day until the end of the differentiation with 20 ng/mL HGF, 0.05 nM dexamethasone (Sigma, St. Louis, MO, USA), 10 ng/mL oncostatin M (Peprotech, Cranbury, NJ, USA), 0.25 nM Compound E (Santa Cruz, Dallas, TX, USA), 2.5 nM SB431452 (Tocris, Bristol, UK) and 10 ng/mL vitamin K1 (Roche, Basel, Switzerland).

NSCs in 2D and the corresponding cells included and printed in the bioink were cultivated in appropriate media for the differentiation first in MNPs and then in MNs (Figure 6).
NSCs were plated on a vitronectin-coated plate for the 2D condition, or printed in the bioink for the 3D condition, and cultivated for 7 days with the MNP Differentiation Medium, composed of Neurobasal 2X, Advanced DMEM/F12 2X, Neural induction supplement 50X, 0.1 µM Retinoic Acid (Sigma-Aldrich, Milan, Italy) and 0.5 µM Purmorphamine (ThermoFisher Scientific Inc., USA).
MNPs were cultivated for 7 days in a medium composed of Neurobasal 2X, Advanced DMEM/F12 2X, 0.5 µM Retinoic Acid, 0.1 µM Purmorphamine, 10 ng/mL GDNF (ThermoFisher Scientific Inc., USA), 10ng/mL IGF (ThermoFisher Scientific Inc., USA) and 10 ng/mL BDNF (ThermoFisher Scientific Inc., Waltham, MA, USA) to obtain iMNs. iMNs were then cultivated for 7 days in the same medium of MNPs with the addition of 0.1 µM Compound E (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for the complete differentiation in mMNs, as previously described [51 (link)].

Aβ40 and Aβ42 levels were measured by enzyme-linked immunoabsorbent assay (ELISA) using conditioned medium collected from cultured neurospheres isolated from Non-Tg and TgCRND8 mice. The ELISA (Biosource International) was performed as previously described (Pardossi-Piquard et al., 2009 (link)). For Aβ quantification, neurosphere media was conditioned for 4 days with samples taken from paired cultures (Non-Tg and TgCRND8) between passages 3 and 7. To assess the effects of γ-secretase inhibition, cells were treated overnight with 100 nM Compound E (Santa Cruz) and dimethyl sulfoxide (DMSO) was used as a negative control as previously described (Beher et al., 2001 (link)).