Embryonic stem cell ERK, AKT, plus STAT3 response dynamics combinatorics are heterogeneous but NANOG state independent

Signaling is central in cell fate regulation, and relevant information is encoded in its activity over time (i.e., dynamics). However, simultaneous dynamics quantification of several pathways in single mammalian stem cells has not yet been accomplished. Here we generate mouse embryonic stem cell (ESC) lines simultaneously expressing fluorescent reporters for ERK, AKT, and STAT3 signaling activity, which all control pluripotency. We quantify their single-cell dynamics combinations in response to different self-renewal stimuli and find striking heterogeneity for all pathways, some dependent on cell cycle but not pluripotency states, even in ESC populations currently assumed to be highly homogeneous. Pathways are mostly independently regulated, but some context-dependent correlations exist. These quantifications reveal surprising single-cell heterogeneity in the important cell fate control layer of signaling dynamics combinations and raise fundamental questions about the role of signaling in (stem) cell fate control.

Automated summary

Signaling dynamics play a central role in cell fate regulation, but quantifying multiple pathways simultaneously in single mammalian stem cells has not been achieved. In this study, fluorescent reporters for ERK, AKT, and STAT3 signaling activity were generated in mouse embryonic stem cell (ESC) lines, and their single-cell dynamics combinations were quantified. Heterogeneity was observed for all pathways, with some dependence on cell cycle but not pluripotency states, even in presumed homogeneous ESC populations. The pathways are mostly independently regulated, but context-dependent correlations exist. These findings reveal surprising single-cell heterogeneity in the control of cell fate by signaling dynamics and raise fundamental questions about its role in (stem) cell fate control.