Maintenance of human embryonic stem cells (hESCs)

All human pluripotent stem cell studies were carried out in accordance with approval from the National University of Singapore’s Institutional Review Board (IRB 12–451). Pluripotent hESCs H1 (karyotype: 46, XY; WiCell Research Institute, WA01, RRID:CVCL_9771) were maintained on 10 μg/ml LN521 (Biolamina AB, LN521)-coated culture plates with daily change of Nutristem® (Biological Industries, 05-100-1 A) medium. Routine monitoring of pluripotent markers POU5F1 (Santa Cruz, sc-5279, RRID:AB_628051) and Tra1–60 (Millipore, MAB4360, RRID:AB_2119183) by flow cytometry and genomic stability by karyotyping were performed at the Singapore General Hospital cytogenetics laboratory. Cells were split at 200,000 cells per well and were passaged at 80% confluence by gentle dissociation with TrypLE (ThermoFisher, 12563011) at 37 °C for 8 mins to dissociate the single cells. The cell suspension was then collected and centrifuged at 800 rpm for 4 mins. Supernatants were discarded and the cell pellets were resuspended in 1 mL of pre-warm Nutristem® medium. Bright-field images were taken with a Leica microscope.