Geltrex

Human embryonic stem cell line H9 (WA09, WiCell; NIH registration number: 0062) was cultured in a standard culture system using mTeSR1 medium (Stemcell Technologies) and lactate dehydrogenase-elevating virus (LDEV)-free human embryonic stem cell qualified reduced growth factor basement membrane matrix Geltrex (Thermo Fisher Scientific) per manufacturer instruction. The cell line was test negative for mycoplasma contamination (LookOut Mycoplasma PCR Detection Kit, Sigma-Aldrich). hESCs were seeded as single cells on glass bottom dishes (MatTek Corporation) coated with 1% (v/v) Geltrex at a density of 20,000 cells cm^-1 with ROCK inhibitor Y27632 (10 µM; Tocris). 24 h after cell seeding, cell culture medium was replaced with fresh mTeSR1 medium without Y27632. Sonoporation experiments were conducted one day after cell seeding (day 1). For other experiments, hESCs were cultured in mTeSR1 medium up to day 8, without losing pluripotency. Culture medium was replenished daily.

Cells were incubated at 37°C in 5% CO₂. Human induced PSCs (hiPSCs) were grown and expanded under feeder-free conditions using Essential 8 medium (E8; Life Technologies) on six-well plates coated with Geltrex (Invitrogen) at a concentration of 1:100. The medium was replaced daily and cells were passaged at 70% confluency via 5–10 min of 37°C incubation with Versene solution (0.48 mM) (Life Technologies) to detach clumps of cells. Cell clumps were resuspended at a ratio of 1:6–1:12 in E8 with 10 μM ROCK inhibitor (Y-27632 dihydrochloride; Tocris) and seeded in fresh Geltrex-coated six-well plates. For differentiation, hiPSCs were grown to 90%–100% confluency.