2.2. Maintenance of iPSCs

All cells were regularly tested for mycoplasma and certified mycoplasma free. All iPSCs were maintained in StemFlex™ Medium (Thermo Fisher, Cat# A3349401) without antibiotics at 37°C, 5% CO₂, 5% O₂ conditions using sterile six‐well NUNC™ plates (Thermo Fisher, Cat# 140675) coated with Geltrex™ (Thermo Fisher, Cat# A1413302). Geltrex™ solution was prepared in 2–4°C Dulbecco's Modified Eagle's Medium/Nutrient Mixture F‐12 Ham (DMEM/F12) (Sigma Aldrich, Cat# D6421) according to the manufacturer's instructions, and all plates were coated 1 h before use.

Maintenance passaging of iPSCs was done with Versene‐ethylenediaminetetraacetic acid (EDTA) solution (Lonza, Cat# BE17‐711 E) according to the manufacturer's instructions. Cells at 70%–80% confluence were washed with 1 ml/well room temperature Hank's Balanced Salt Solution (HBSS) (Thermo Fisher, Cat# 14170‐161) and incubated with 1 ml/well room temperature Versene for 3–4 min. After aspirating Versene from each well, cells were detached gently (2–3 wells at a time) with 5–6 ml of room temperature StemFlex™, using 5 ml or 10 ml stripettes. Detached cells were collected in new 50 ml conical tube(s) and were gently broken down with 5 ml or 10 ml stripettes until the size of cell clumps were reduced to that of “dots” when viewed with unaided eyes. Passaging ratio was kept between 1:6 and 1:18 depending on the experimental requirements. Spontaneously differentiated iPSC colonies were regularly cleaned prior to passaging with sterile aspirator pipettes with 10/20 μl pipette tips inserted at the end. Time spent outside the incubator for cleaning was always kept under 5 min.