Established iPSC lines were cultured in hESC medium without P/S for 3 days without refreshing medium. Spent medium was subjected to the MycoAlert Mycoplasma Detection Kit (Lonza) for the presence of mycoplasma species. For flow cytometric analysis of cell surface pluripotency markers SSEA4 and Tra-1-60, iPSCs were dissociated into single cells with Accutase (Innovative Cell Technologies) and incubated with Alexa Fluor 647 anti-human SSEA-4 (BioLegend) and PE anti-human Tra-1-60 (BD Biosciences) antibodies. Samples were analyzed using the BD FACSCalibur system (BD Biosciences). Cells were plotted according to forward scatter and side scatter profiles and gated to exclude cell doublets and debris. Data were analyzed with FlowJo software (FlowJo). Table S1 includes the proportion of double-positive cells for each iPSC line. For confirmation of loss of SeV reprogramming vector expression, total RNA was isolated from iPSC lines cultured under feeder-free conditions using the miRNeasy Mini Kit (QIAGEN) and quantified with the RNA 6000 Nano Kit (Agilent) on the Agilent Bioanalyzer system. 1 μg of RNA was used for cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
