Multidish

As previously reported [6 (link)], to induce oligodendrocyte differentiation, rabbit PSCs were digested with trypsin, suspended in embryoid body (EB) medium containing 78% DMEM/Ham’s F-12 supplemented with 20% KSR, 1% nonessential amino acids, 50 units/ml penicillin, 50 μg/ml streptomycin, 0.1 mM β-mercaptoethanol, 1% N-2 supplement (Invitrogen), 4 μM all-trans-retinoic acid (Sigma-Aldrich) and 10 μM SB431542 (Tocris Bioscience, Bristol, UK). To achieve single EBs of a uniform size, 1,000 PSCs in a volume of 100 μl were dispensed into each well of low-cell-adhesion 96-well round bottom plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA), tapped gently and cultured at 37 C under 6% CO₂ in air. To obtain differentiated oligodendrocytes, six EBs were transferred to a Matrigel (BD Bioscience, Franklin Lakes, NJ, USA)-coated 4-well Multidish (Nunc) and allowed to attach to the bottoms of the wells. The medium
was then changed from EB medium to neural differentiation medium (the same formulation as EB medium, with the addition of 10% KSR). After 10 days, the medium was changed from neural differentiation medium to the culture medium without retinoic acid or SB431542 but with 100 ng/ml Noggin (Wako). The cells were cultured for a further 20 days, with fresh medium added every day.