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

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), 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. The 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 trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM. ATAC-seq for wt XEN cells and XEN cells after KO of GATA4 or GATA6

哺乳动物胚胎中，多能上胚层（pluripotent epiblast, EPI）与原始（胚外）内胚层（primitive (extra-embryonic) endoderm, PrE）这两种截然不同的细胞命运，均源自共同的祖细胞——内细胞团（inner cell mass, ICM）。为探究这两种姊妹细胞命运的构建机制，我们利用了胚胎干细胞（embryonic stem, ES）与胚外内胚层干细胞（extraembryonic endoderm, XEN）——二者分别对应体内EPI与PrE的体外培养模型。通过ES与XEN细胞间的双向重编程，结合单细胞RNA测序与转座酶可及性染色质测序（ATAC-seq）分析，我们揭示了细胞状态转换的不同速率、效率与轨迹，并鉴定出互转过程中的驱动因子与阻碍因素。尽管GATA4介导的ES向诱导型XEN（iXEN）转化快速且近乎完全确定，但OCT4、KLF4与SOX2诱导的XEN向诱导多能干细胞（iPS）重编程，其效率与动力学均有所下降。由Gata4维持的主导性PrE转录程序，以及EPI细胞整体升高的染色质可及性，凸显了两种细胞状态的可塑性差异。将体外重编程轨迹映射至体内胚胎时发现，任一方向的重编程均沿体内EPI与PrE状态进行，并在二者间切换，无需经过ICM阶段。我们对野生型XEN细胞，以及敲除GATA4或GATA6后的XEN细胞进行了ATAC-seq分析。