Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency

Abstract

Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

“…NANOG is the pluripotency TF for which a direct quantitative correlation between levels of expression and ESC self‐renewal efficiency has been most clearly demonstrated (Chambers et al , 2003, 2007; Abranches et al , 2014). Accordingly, NANOG is considered to act as a rheostat to tune ESC self‐renewal efficiency (Mullin & Chambers, 2012).…”

“…In contrast to reduced NANOG expression, which is a reversible state (Chambers et al , 2007; Abranches et al , 2014; Filipczyk et al , 2015), loss of ESRRB commits cells to exit the naïve state. This can be clearly seen in clonal self‐renewal assays where Esrrb negative cells are essentially unable to form colonies in LIF.…”

“…The activity of a relatively well‐characterized pluripotency gene regulatory network, centered on the triumvirate OCT4, SOX2 and NANOG, controls the dual abilities of ESCs to self‐renew and to differentiate (Jaenisch & Young, 2008; Chambers & Tomlinson, 2009; Ng & Surani, 2011). While populations of ESCs cultured in LIF/FCS express relatively homogeneous levels of Oct4 and Sox2, individual ESCs show varying levels of Nanog expression (Chambers et al , 2007; Kalmar et al , 2009; Abranches et al , 2014). Heterogeneous expression has also been reported for Esrrb (van den Berg et al , 2008), Klf4 (Niwa et al , 2009), Tbx3 (Niwa et al , 2009; Russell et al , 2015), Rex1 (Toyooka et al , 2008), and Stella (Hayashi et al , 2008).…”

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

“…For Nanog, expression is bimodal, with high and low local maxima (Chambers et al, 2007). Nanog expression relates to phenotypic behaviour, with low-expressing cells showing a tendency to differentiate and high-expressing cells tending towards self-renewal (Chambers et al, 2007;Abranches et al, 2014). Treatment of ESCs with 2i inhibitors (Ying et al, 2008) favours self-renewal, and shifts Nanog expression towards a unimodal high distribution.…”

Single Cell Analysis Reveals Concomitant Transcription of Pluripotent and Lineage Markers During the Early Steps of Differentiation of Embryonic Stem Cells

“…This variability can have functional consequences, as shown for instance by the finding that cells expressing low levels of the Nanog gene are more prone to differentiate than their Nanog high counterparts [6]. Recently, Nanog low cells were found to express lineage-associated markers, but no coherent pattern of lineage priming could be identified in these cells [7]. The observation of inherently dynamic gene expression in ESCs has led to the view that pluripotency is a property of stem cell populations rather than individual cells, and that the cells within such populations transit stochastically between distinct cellular states [8,9].…”