hiPSCs with W101C KCNJ5 variant were cultured in FTDA medium (DMEM/F12 (Gibco, Life Technologies, Paisley, UK) with 2 mM L-Glutamine (ThermoFisher), 1× ITS (Corning, Bedford, MA, USA), 0.1% HSA (Lucerna-Chem, Lucerne, Switzerland), CD lipid concentrate (Gibco, Life Technologies, Paisley, UK), 50 nM Dorsomorphin (Santa Cruz Biotechnology, Heidelberg, Germany), 2.5 ng/mL Activin A (STEMCELL Technologies, Vancouver, BC, Canada), 0.5 ng/mL TGFß1 (ThermoFisher), 30 ng/mL FGF2 (PeproTech, Cranbury, NJ, USA)) on Matrigel (Corning)- or Geltrex (Thermo Fisher Scientific)-coated plates [60 (link),61 (link)]. Cells were examined daily under a light microscope for differentiated sites, density, and morphology.
At 90–100% confluence of the monolayer, hiPSC were passaged with Accutase (Sigma-Aldrich, Taufkirchen, Germany) and 10 µM Y-27632 (Abcam, Amsterdam, Netherlands). For maintenance, 500,000 to 600,000 cells per 6-well plate were laid out in new FTDA medium with ROCK-inhibitor.
At 90–100% confluence of the monolayer, hiPSC were passaged with Accutase (Sigma-Aldrich, Taufkirchen, Germany) and 10 µM Y-27632 (Abcam, Amsterdam, Netherlands). For maintenance, 500,000 to 600,000 cells per 6-well plate were laid out in new FTDA medium with ROCK-inhibitor.
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