The low level of transcriptional differences between undifferentiated hESCs and hiPSCs does not exclude the existence of iPSC-specific aberrations that become detectable only after differentiation. We performed RNA-sequencing of fibroblast-like cells derived from 8 hESC subclones (2 hESC SC and 6 hESC GFP lines) and 6 hiPSC subclones using the same in vitro differentiation protocol as described above (Fig. 1A). Only two genes were consistently upregulated in hiPSC-derived fibroblast-like cells compared to hESC-derived fibroblast-like cells from both genetic backgrounds, and they did not overlap with the 49 DEGs between undifferentiated hESC and hiPSC lines (Supplementary Fig. 3A,B). However, HUES2-derived fibroblast-like cells tended to cluster together and apart from HUES3-derived fibroblast-like cells using PCA analysis (Supplementary Fig. 1B), which is consistent with the segregation of undifferentiated cells by genetic background. We infer that genetic background also drives transcriptional variation in differentiated cell populations, and that any transcriptional differences observed between undifferentiated hESC and hiPSC lines do not persist in differentiated fibroblast-like cells.
