Ultra low non adherent 6 well plates

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.

Neuroprogenitor cells were selectively expanded using non-adherent culture [16 (link),17 (link),18 (link),19 (link),20 ,21 (link)]. Briefly, aMSCs were seeded at 10,000 cells/cm² density onto Ultra Low^® non-adherent 6-well plates (Corning, Corning, NY, USA) in sphere-forming medium (SFM) comprising DMEM/F12 (Thermo Fisher Scientific) supplemented with 2% B27 (Thermo Fisher Scientific), bFGF, and EGF (20 ng/mL, Peprotech, Rehovot, Israel). Cultures were maintained for 8–10 days with medium refreshed every 48 h. At timed intervals, selected spheres were partially dissociated via incubation in TrypLE Express for efficient cell counting and determination of proportion of cells immunopositive for the neural stem/progenitor marker nestin [24 (link)] and the OL lineage determining factor Olig2 [25 (link),26 (link)].