Feeder-free culture of hESCs and iPSCs

hESCs [WA09 (H9); WiCell Research Institute], BJ-iPSCs [38 (link)], and CHCHD2 KO/reconstitution subclones were maintained in mTeSR1 (Stem Cell Technology, #85,851) or MACS-iPSC brew medium (Milltany Biotechnology, #130-104-368) on plates coated with Matrigel (Corning, #354,277) diluted at 1:80 in hESC basal medium (DMEM/F12 supplemented with 1% non-essential amino acids, 0.1% β-mercaptoethanol, and 0.1% gentamicin, Gibco) for feeder-free conditions. Cells were incubated in 37 °C, 5% CO₂ conditions. The medium was replaced every day up to passaging, and the cells were enzymatically dissociated using a dispase solution (Gibco, #17,105,041) with 10 μM of Y-27632 (Biogems, #1,293,823). 1 ~ 2 × 10⁵ cells were seeding in 60Ø plate for transfer. 4 h mitomycin C-treated mouse embryonic fibroblast (MEF) was used as feeder cells for H9 feeder culture. Inverted microscope [Olympus, CKX-41 (Light source: 6 V/30W halogen lamp, Software: ProgRes Capture Pro)] was used to capture images for live cells.