Defining reprogramming checkpoints from single-cell analysis of induced pluripotency

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a novel combination of epigenetic and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress towards iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play novel functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenetic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.