Culturing and Transfecting Human iPSCs and HEK293T Cells

HEK293T cells were purchased from ATCC (CRL-3216) and cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep at 37 °C, 5% CO₂. Human iPS cell line HMGUi001-A2 (sex: female), a sub-clone of HMGUi001-A^{57 (link)}, was kindly provided by Dr Kühn (MDC) and authenticated by karyotyping. Human iPSCs were maintained on Geltrex-coated (Invitrogen) plates in E8 media (STEMCELL Technologies). The medium was changed daily, and cells were passaged every ∼3 days as cell clumps or single cells using 0.5 mM EDTA (Invitrogen) or Accutase (Invitrogen), respectively. Medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Sigma) when iPSCs were thawed or passaged as single cells. All cell lines tested negative for mycoplasma contamination which was carried out routinely.
For transient transfection experiments in HEK293T cells were transfected using Polyethylenimine “Max” (PEI) (Polysciences) in OptiMEM medium (Invitrogen).