A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming. [Display omitted] ► Transcriptional analysis of iPSC formation reveals biphasic process ► c-Myc/Klf4 drive first phase whereas Oct4/Sox2/Klf4 drive second phase ► Bivalent genes form gradually whereas DNA methylation changes occur late ► Refractory cells can be rescued by elevated reprogramming factor expression By sorting fibroblasts induced to reprogram to iPSCs, the molecular characteristics of cells in the early and late stages of reprogramming are revealed, as well as the attributes of the cells refractory to reprogramming. New genes that enhance reprogramming efficiency are identified.