Random Differentiation of H9 Cells

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.