Optimal Stem Cell Aggregation for Differentiation

Stem cells were routinely maintained in either mTeSR1 or Essential 8 (E8). However, for all cell aggregation experiments, cells were maintained on 1% (vol/vol) Matrigel-GFR™ (BD Biosciences) coated dishes at 37 °C under hypoxic conditions (10% CO₂/5%O₂) in mTeSR1 (Stem Cell Technologies) prior to reaggregation^{28 (link), 51 (link), 71 (link)}. Cells were passaged with Accutase (Sigma) for 8–10 minutes, dissociated to single cells, quenched with mTeSR1 plus 5 μM blebbistatin (B; Sigma), pelleted at 80 × g for 5 minutes, resuspended in mTeSR1 + B and plated at 5,000 cells per 35 mm well^{32 (link)}. After 48 hours, cells were fed without B. To minimize cell stress, no antibiotics were used^{52 (link)}.