Human embryonic stem cell (hESC) cultures
Undifferentiated hESCs of SA02 (Sahlgrenska 2) line (Cellartis AB, Göteborg, Sweden; see NIH Human Embryonic Stem Cell Registry at [46]) were maintained over a monolayer of human "feeder cells" (hFCs; human foreskin fibroblasts, ATCC; cell line CCD-1112Sk). Feeder cells were grown in hFC medium (Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% heat-inactivated FCS (Stem Cell Technologies, USA) and 0.5% Penicillin/Streptomycin mix) for 11 passages. One day prior to hESC plating, hFC medium was washed away from the hFCs, the latter were resuspended in a hESC proliferation medium (VitroHES media (Vitrolife AB, Sweden) supplemented with 4 ng/ml human recombinant basic FGF (hrbFGF, Biosource International, USA) and plated in a central ring of gelatinized in vitro fertilization (IVF) dishes with a cell density of 120,000 cells/dish. The outer rings of the IVF dishes were filled with Dulbecco's modified Eagle medium (DMEM) supplemented with 0.5% Penicillin/Streptomycin mix. One half of the culture medium was replaced every other day. The cells were maintained at 37°C, 5% CO2, 95% humidity settings. Every 6 days, fragments of the hESC colonies (around 10–14 colonies per dish, measuring around 0.015 × 0.015 mm) that had an unaltered morphology (indicating lack of spontaneous differentiation) were mechanically cut from dishes using stem cell knives/transfer pipettes (SweMed Lab International AB, Sweden) and then plated on fresh hFCs.