Neuronal Spheroid Formation from hBM-MSCs

To obtain neuronal spheroids, hBM-MSCs were cultured in a low-attachment dish (Corning) with serum-free sphere-forming medium comprising advanced DMEM/F-12 (1:1) (Gibco) supplemented with 0.5 mM l-glutamine, N2 supplement (Gibco), 20 ng/mL recombinant human EGF (Wako), 20 ng/mL human FGF-2 (Wako), and 2% B27 (Gibco). Following the formation of spheroids, neuronal spheroids obtained using low-attachment dishes or MSC spheroids obtained under shaking-culture conditions were seeded on fibronectin (Wako)-coated 6-well chambers (Matsunami Glass Ind. Inc., Kishiwada, Osaka, Japan) using induction medium comprising advanced DMEM/F12 (1:1) (Gibco) supplemented with 10% FBS (Hyclone, GE Healthcare), N2 supplement (Gibco), 1% P/S (Wako), and 10 mM HEPES (Dojindo Molecular Technologies, Inc.) (Morikawa et al., 2009b (link); Choi et al., 2017 (link)). The medium was changed every 3–4 days until day 10.