Fibroblasts were transduced in one well of a six-well plate using Sendai vectors expressing hOCT3/4, hSOX2, hKLF4, and hc-MYC 50 (CytoTune™, Life Technologies, Cat. no. A1377801). The transduced cells were cultured on an irradiated mouse embryonic fibroblast (MEF-CF1) feeder layer on a 10 cm2 tissue culture dish in iPSC medium consisting of Advanced DMEM (Life technologies, UK) supplemented with 10% Knockout Serum Replacement (KOSR, Life technologies, UK), 2 mM L-glutamine (Life technologies, UK) 0.007% 2-mercaptoethanol (Sigma-Aldrich, UK), 4 ng/mL of recombinant Zebrafish Fibroblast Growth Factor-2 (CSCR, University of Cambridge), and 1% Pen/Strep (Life technologies, UK). Cells with an iPSC morphology appeared approximately 25 to 30 days post-transduction. The undifferentiated colonies (six per donor) were picked between days 30-40, transferred onto 12-well MEF-CF1 feeder plates and cultured in iPSC medium with daily media change until ready to passage. Cells were passaged every five to seven days, depending on the confluence and morphology of the cells, at a maximum 1:3 split ratio until established – usually at passage five or six. Once the iPSC lines were established in culture, three of the six lines were selected based on morphological qualities (undifferentiated, roundness and compactness of colonies) and expanded for banking and characterisation.
