Selected iPSC lines were assessed for their pluripotency and differentiation properties by culturing the cells under conditions favouring the formation of the three embryonic germ layers, and subsequent immunostaining with markers specific for pluripotency and differentiation. Differentiation was performed as described previously 58. Briefly, iPSCs grown in feeder-dependent or feeder-free conditions were harvested using either collagenase and dispase or EDTA, respectively. Colonies were collected, washed in media and mechanically broken up before being re-plated onto 24-well mouse embryonic fibroblast (MEF) feeder plates or pre-coated gelatine/FBS plates. For pluripotency assays, feeder-dependent colonies were seeded on MEF feeder plates and feeder-free colonies onto Vitronectin plates. For the differentiation assay, colonies were grown on gelatine/FBS plates. Prior to differentiation to mesoderm (dME), endoderm (dEN), and neuroectoderm (dEC), cells were cultured overnight in pre-differentiation media CDM-PVA supplemented with recombinant Activin-A (10 ng/ml; CSCR, University of Cambridge) and zebrafish FGF2 (12 ng/ml; CSCR, University of Cambridge).
