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. Contradicting this, naive PSC grown in fully defined ‘2i’ environmental conditions, containing small molecule inhibitors of MEK and GSK3, show homogenous pluripotency and lineage marker expression. Here we report that 2i induces greater genome-wide heterogeneity than traditional serum-containing growth conditions at the population level across both male and female PSCs. Heterogeneity is dynamic and reversible over time, consistent with a dynamic metastable equilibrium of the pluripotent state. We further show that 2i conditions increase heterogeneity specifically in the calcium signalling pathway at both the population and single-cell level. This is driven by loss of robustness regulators in the form of negative feedback to the upstream EGF receptor. Our findings show that metastability occurs in both 2i and serum PSCs which has implications for our understanding of the nature of the pluripotent state and 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, with induced heterogeneity potentially inducing cancer metastasis and drug resistance. Pluripotent stem cell lines including embryonic stem cells and embryonic germ cells were grown in either serum (FCS) or 2i conditons. Transcriptomes of 22 samples (9 in serum and 13 in 2i) were profiled in batch 1, including 10 female and 12 male cell lines. Transcriptomes of 18 samples (9 in serum and 9 in 2i) were profiled in batch 2 across passages 11, 14 and 17 for three cell lines for each growth condition. All cell lines were derived from Oct4ΔPE-GFP transgenic male mice crossed with with 129SvEv female mice.