▲ CRITICAL hESCs and human induced pluripotent stem cells can vary widely in their transfection efficiency, tolerance of single-cell dissociation and maintenance conditions. For a given cell line of interest, relevant literature or the distributor should be consulted. 30Maintaining HUES9 cells. We routinely maintain HUES9 cells (a hESC cell line) in feeder-free conditions with mTesR1 medium. Prepare mTeSR1 medium by adding the 5× supplement included with the basal medium and 100 μg ml–1 Normocin.31Prepare a 10-ml aliquot of mTeSR1 medium supplemented further with 10 μM ROCK inhibitor.32Coating a tissue culture plate. Dilute cold GelTrex 1:100 in cold DMEM and coat the entire surface of a 100-mm tissue culture plate.33Place the plate in an incubator for at least 30 min at 37 °C.34Thaw a vial of cells at 37 °C, transfer the cells to a 15-ml Falcon tube, add 5 ml of mTeSR1 medium and pellet at 200g for 5 min at room temperature.35Aspirate the GelTrex coating (Step 32) and seed ~1 × 106 cells with 10 ml of mTeSR1 medium containing ROCK inhibitor from Step 31.36Replace with mTeSR1 medium without
