First we studied whether the SeV reprogramming method affects global transcription. The parental hESC subclones were infected with GFP-expressing SeV (SeV-GFP) and passaged until GFP fluorescence was no longer detectable before analyzing cell lines by RNA-sequencing (Fig. 1A and 2A). We found a common set of 63 genes that was differentially expressed between three uninfected hESC subclones (hESC SCs) and three SeV-GFP infected hESC subclones (hESC GFPs) from each genetic background, which demonstrates that viral infection itself leads to subtle but statistically significant transcriptional changes that persist after viral loss (Fig. 2B). This 63-DEG set consistently separated hESC SC lines from hESC GFP lines (Fig. 2C). Gene Ontology terms significantly enriched among these 63 DEGs are related to transcription, DNA binding, and development (Supplementary Fig. 1E). Based on these observations, we decided to use expression data from hESC GFP lines as controls for all subsequent comparisons with SeV-generated hiPSC lines.
