Signalling pathways drive heterogeneity of ground state pluripotency

Pluripotent stem cells (PSCs) can self-renew indefinitely while maintaining the ability to generate all cell types of the body. This plasticity is proposed to require heterogeneity in gene expression, driving a metastable state which may allow flexible cell fate choices. Contrary to this, naive PSC grown in fully defined 92i9 environmental conditions, containing small molecule inhibitors of MEK and GSK3 kinases, show homogenous pluripotency and lineage marker expression. However, here we show that 2i induces greater genome-wide heterogeneity than traditional serum-containing growth environments at the population level across both male and female PSCs. This heterogeneity is dynamic and reversible over time, consistent with a dynamic metastable equilibrium of the pluripotent state. We further show that the 2i environment causes increased heterogeneity in the calcium signalling pathway at both the population and single-cell level. Mechanistically, we identify loss of robustness regulators in the form of negative feedback to the upstream EGF receptor. Our findings advance the current understanding of the plastic nature of the pluripotent state and highlight the role of signalling pathways in the control of transcriptional heterogeneity. Furthermore, our results have critical implications for the current use of kinase inhibitors in the clinic, where inducing heterogeneity may increase the risk of cancer metastasis and drug resistance.