Abstract
BackgroundThe pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells’ ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state.Methodology/Principal FindingsWe describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment.Conclusions/SignificanceThe novel reporter ES cell line here described allows an accurate monitoring of Nanog’s dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape.
Highlights
Embryonic Stem (ES) cells are characterized by their selfrenewal capacity and pluripotenciality [1,2]
Generation of a Nanog:VNP Reporter embryonic stem (ES) Cell Line To monitor the dynamic Nanog expression in ES cells, we have generated a novel ES cell line containing a transgenic Bacterial Artificial Chromosome (BAC) with a fluorescent reporter inserted under the control of the Nanog regulatory regions
The recombined BAC was electroporated into E14tg2a ES cells, and neomycin selection was applied to select clones with BAC transgene integration
Summary
Embryonic Stem (ES) cells are characterized by their selfrenewal capacity and pluripotenciality [1,2]. Extensive characterization of the transcriptional program elicited by these three TFs revealed that they function in concert to sustain the ES cell state by activating other pluripotency genes while, simultaneously, repressing differentiation-promoting genes [7,9,10] This repression is thought to play a central role in maintaining the pluripotent state, reducing its vulnerability to the myriad of extrinsic signals that promote differentiation along the various embryonic lineages. Recent work has shown that both Oct and Sox can function as lineage specifiers, assisting the emergence of mesendodermal and neuroectodermal fates, respectively [11,12] These findings support a different view of the pluripotent state, as a highly unstable and transient cellular state, driven by the competing lineage-promoting activities of the different ‘‘pluripotency’’ factors [13], instead of a ground state implemented and maintained by the NOS circuitry. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is critical to develop a better understanding of the pluripotent state
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