Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities [scRNA-Seq]

Two distinct lineages, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common inner cell mass (ICM) progenitor cells in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells – in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. A dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro to embryo trajectories revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM. Reprogramming XEN-to-iPS and ES-to-iXEN cells were assayed for gene expression using single-cell RNA-seq. Given the low proportions of cells undergoing lineage conversion (based on marker expression) from XEN to iPS cells at all timepoints assessed, and ES cells at the 9h timepoint, subpopulations were isolated using fluorescence-activated cell sorting (FACS) and reconstituted at roughly equal proportions. This serves to: (1) retain all the heterogeneity within the reprogramming population, and (2) enrich for cells undergoing key lineage marker expression changes. In addition to reprogramming stages, cells at starting and final states were also sampled.