Directed Differentiation of Human iPSCs and ESCs

Human iPSCs and ESCs maintained on MEFs were passaged on Matrigel (GFR, BD Biosciences) thin coated 6-well plates (8.7 ug/cm²) and cultured in mTeSR1 medium (WiCell Institute) for 5–6 days to deplete the feeder cells. Cells were washed with PBS and incubated with 1 ml/well Versene solution (Invitrogen) at 37°C for 5 minutes to singularize the cells and seeded on Matrigel coated plate at the density of 100,000 cells/cm² in mTeSR1 medium supplemented with 10 μM ROCK inhibitor (Y-27632, CalBiochem). The medium was changed daily, and after 3–4 days when the monolayer of cells reached 80–90% confluence, a thin layer of Matrigel was overlaid by freshly mixing Matrigel (GFR), 0.5 mg (faster growing lines, i.e. DF19-9-11T, DF19-9-7T, IMR90 C4 or H9) or 1 mg (slower growing lines, i.e. DF6-9-9T, H1), in 15 ml ice cold mTeSR1 medium and replacing the medium in each well of a 6-well plate with 2.5 ml of Matrigel containing mTeSR1. Cells were cultured in mTeSR1 medium for another 1–2 days until the cells were 100% confluent, which is referred to as day 0 when the medium was replaced with 2.5 ml of RPMI 1640 basal medium (Invitrogen) plus B-27 without insulin supplement (Invitrogen) containing Activin A (100 ng/ml, R&D Systems) and Matrigel (0.5 mg Matrigel/6-well plate). After 24 hours, the medium was changed with the same medium as day 0 (3 ml/well) without Matrigel but supplemented with BMP4 (5–10 ng/ml, R&D Systems) and bFGF (5–10 ng/ml, Invitrogen) for another 4 days without medium change. At day 5, the medium was changed to RPMI plus B27 complete supplement (Invitrogen), and the medium was changed every 2–3 days. For H9 cells, 1% KnockOut serum replacer (Invitrogen) was added at day 0 in a subset of experiments.