Culture of BMSCs was performed in accordance with our previous publication [8 (link)]. Briefly, reamed human bone marrow was resuspended in growth medium containing αMEM supplemented with 15% foetal bovine serum (FBS) and seeded on tissue culture plates. Non-adherent cells were removed following medium exchange 48 h later. BMSCs were maintained in growth medium until reaching 80% confluency, then detached with TrypLE Express and passaged in a 1:2 ratio. For neurosphere formation, Passage 5 (P5) BMSCs were seeded at a density of 50,000 cells per well onto Ultra Low® non-adherent 6-well plates (Corning) in sphere-forming medium comprising of neurobasal medium supplemented with 2% B27, 1% GlutaMAX, 20 ng/ml bFGF2 and 20 ng/ml EGF. To bias differentiation, 1 μM SB4 was added to sphere-forming medium in the SB4 treatment group. Neurospheres were maintained for 8 days with culture medium exchanged every 3 days.
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