Identification of novel embryonic differentiation stage by combined cell tracking and high dimensional protein imaging

Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we tracked individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm lineage commitment. Unexpectedly, we could identify a novel developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo as well. RNASeq revealed cells co-expressing SOX1 and FOXA2 to have a unique cells state characterised by expression of both endoderm as well as neuroectoderm genes with lineage potential towards both germ layers. Overall design: To better characterize the Sox1 and FoxA2 co-expressing cells (markers of neuroectoderm and endoderm respectively) discovered from proteomincs experiments as well as the lineage portential of their progeny, we generated a double reporter mouse embryonic stem cells line Sox1-EGFP/FoxA2mCherry. These cells underwent Retinoic Acid (RA) differentiation for 2-,4- or 6 days, were FACS sorted based on reporter expression into populations of interest and underwent bulk RNASeq. In addition, Sox1-FoxA2+, Sox1+FoxA2- and Sox1+FoxA2+ cells were isolated by FACS sorting after 2 days of RA diffentiation, replated and allowed to grow for an additional 2- or 4- days in RA medium prior to RNA extraction. These samples are marked with 'Progeny' in the sample name