First, we subcloned each line in order to ensure genetic and epigenetic homogeneity of cells and to properly control for the clonal origin of hiPSCs (Fig. 1A). We differentiated one hESC subclone from each background by switching cells to serum-containing medium without basic fibroblast growth factor (bFGF), which is critical for the maintenance of hESCs, and sorting fibroblast-like cells based on CD90+/TRA-1-81− expression (Fig. 1A,C). These fibroblast-like cells, which resemble primary human fibroblasts by morphological criteria (Fig. 1C), did not form Alkaline Phosphatase (AP)-positive colonies in hESC media, indicating successful differentiation and the absence of residual pluripotent cells in the culture (Supplementary Fig. 1A). Analysis of global gene expression by RNA-sequencing revealed that the fibroblast-like cells were highly similar to dermal fibroblasts but distinct from pluripotent stem cell lines (Supplementary Fig. 1B). Pluripotency-associated promoters, such as POU5F1, LEFTY1, TDGF1, and SCNN1A, were re-methylated and decreased in expression levels whereas fibroblast-specific promoters such as TMEM173, EMILIN1, LMNA, and RIN2 were demethylated and regained expression in fibroblast-like cells (Fig. 1D). In a final step, the fibroblast-like cultures were reprogrammed into hiPSCs by
