Signalling pathways drive heterogeneity of ground state pluripotency

Abstract

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 '2i' 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.RNAs, are not reduced and are instead increased in 2i ( Fig. 2i) arguing against this. This Animal studiesAnimal studies were authorized under a UK Home Office Project License and carried out in a Home Office-designated facility. Cell cultureESCs and EGCs (Oct4ΔPE-GFP) were derived and cultured as previously described 18 .Serum culture conditions contain 15% FCS, 0.1 mM MEM nonessential amino acids, 2 mM lglutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol and 1000U/mL LIF in DMEM-F12 with maintenance on a mouse embryonic fibroblast (MEF) feeder layer. 2i culture conditions contain 1 μ M PD0325901, 3 μ M CHIR99021 and 1000U/mL LIF in N2B27 medium, maintained on laminin (10 μ g/ml). Cells were incubated at 5% CO 2 , 95% relative humidity. All samples were profiled at fewer than 23 passages. For time-course transcriptome profiling EGCs were derived and maintained in either serum or 2i and collected every 3 passages from 11-17. Population transcriptomicsSamples were prepared and processed as described previously. Briefly, 100ng RNA was fragmented, labelled and hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST Arrays.Data was preprocessed in R using robust multiarray averaging, variance stabilising transformation and Combat batch correction 70 . Limma was used for differential expression analysis and for linear regression against cell subtypes, sex and passage 71 . Differential variance was tested using the studentised Breusch-Pagan test with culture condition as the independent variable. Testing statistical enrichment of gene sets was performed using functional annotation ontology 40 ...

Defined conditions for propagation and manipulation of mouse embryonic stem cells

“…We have proposed that the defined culture system using the highly specific 2i inhibitors corrals ES cells in a stable ‘ground state’ (Martello and Smith, 2014). The 2i platform has been widely exploited to study signalling, gene regulation and network control of naive pluripotency, and the transition path from pluripotency to lineage commitment (Blaschke et al, 2013; Carey et al, 2014; Dunn et al, 2014; Ficz et al, 2013; Habibi et al, 2013; Hackett et al, 2017; Hayashi et al, 2012, 2011; Kalkan et al, 2017; Kumar et al, 2014; Leitch et al, 2013a; Marks et al, 2012; McEwen et al, 2018 preprint; Mulas et al, 2017; Murakami et al, 2016; Semrau et al, 2017). Importantly, male ES cells maintained in 2i can retain a euploid karyotype and germline chimaera competency over multiple passages with similar efficiency to cells cultured in serum (Ying et al, 2008; Nichols et al, 2009a; Kiyonari et al, 2010; Leitch et al, 2010, 2013b,a; Leeb et al, 2012; Morgan et al, 2013; Jakubczik et al, 2016; Kalkan et al, 2017; Zhang et al, 2018).…”