Hesc qualified matrix

To remove MEFs, H9 cells from the maintenance culture were transferred on hESC-qualified matrix (BD Biosciences, California, USA)-coated, 60-mm tissue culture plates (Nunc, Langenselbold, Germany) in TESR1 medium (StemCell Technologies) and were maintained for 5 days prior to differentiation. The random differentiation into embryoid bodies (EBs) representing multiple lineages was performed as described previously (Meganathan et al. 2012 (link)). In brief, cell clumps were obtained by cutting and scraping the cells with a passage tool (StemPro EZPassage™ Disposable, Invitrogen) and a cell scraper. On day 0, 80 clumps were seeded in each well of a pluronic-coated, v-bottom plate in 100 µl of random differentiation (RD) medium (DMEM-F12 medium with 20 % KO serum replacement, 1 % non-essential amino acids, penicillin (100 units/ml), streptomycin (100 µg/ml) and 0.1 mM β-mercaptoethanol) containing chemical or vehicle, and the plate was then incubated (37 °C, 5 % CO₂) for 4 days. The EBs were collected on day 4 and were transferred onto a 100-mm bacteriological plate in 15 ml of RD medium containing the chemical or vehicle. The medium was replenished every alternate day until day 14 of differentiation.

To remove the mouse embryonic fibroblasts, the H9 hESCs were transferred from the maintenance culture onto hESC-qualified matrix (BD Biosciences) -coated 60-mm tissue culture plates (Nunc, Langenselbold, Germany) in TESR1 medium (Stem Cell Technologies, Vancouver, BC, Canada). The hiPSCs (foreskin and IMR-90) were maintained on 60-mm tissue culture plates coated with BD Matrigel growth factor reduced in TESR1 medium. Cells were maintained on these plates for 5 days prior to differentiation. The random differentiation of hESCs was performed using the embryoid bodies protocol, as described previously [15 ]. Briefly, the clumps were obtained by cutting and scraping the cells with passage scrapers (StemPro EZPassageTM Disposable; Invitrogen, Carlsbad, CA, USA). On day 0, 100 clumps were seeded in a conical well, coated with Pluronic F-127 (5%) in 100 μl of random differentiation medium (Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium with 20% KO serum replacement, 1% non-essential amino acids, penicillin (100 units/ml), streptomycin (100 μg/ml), 0.1 mM β-mercaptoethanol) containing 1 mM VPA or vehicle, and incubated for 4 days at 37 °C and 5% CO₂. The embryoid bodies were collected on day 4 and transferred onto 100-mm bacteriological plates in 15 ml of random differentiation medium containing 1 mM VPA or vehicle. The medium was replenished every alternate day, until day 14.