Lineage conversion induced by pluripotency factors skips the transient iPSC stage

A combination of Oct4, Sox2, Klf4, and Myc (OSKM) can be utilized to induce all three germ layer cells as well as induced pluripotent stem cells (iPSCs). The three germ layer cells are thought to be originated from transiently formed iPSC-like cells revealed by lineage tracing. However, here we report the two distinct reprogramming trajectories toward iPSCs or induced neural stem cells (iNSCs) from the transcriptional profiles of 33,966 single cells collected at seven sequential time points, induced by OSKM in a monocistronic expression system. The two trajectories are separated at day 6 by lineage-specific environments and not overlapped at later time points. Some cells on the iNSC reprogramming trajectory express Oct4 (Pou5f1) but the lack of co-expression of other iPSC markers, especially Nanog, implies that induction of iNSCs bypasses the iPSC-like state. Detailed trajectory analysis of iNSC reprogramming reveals that the three predicted paths distinguished by endogenous Oct4 expression are largely determined by the expression of exogenous OSKM. This study provides a new insight into OSKM-mediated reprogramming