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 co-occurs in single cells. Sustained pluripotency requires the co-expression of Nanog, Oct4 and Sox2 with specific cohorts of genes. We uncover functional roles for Ehf, Phlda2, and translation initiation factor Eif4a1 for 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. Single cell RNA sequencing of mouse embryonic fibroblast during reprogramming and embryonic stem cells.