Generation and Characterization of a Novel Mouse Embryonic Stem Cell Line with a Dynamic Reporter of Nanog Expression

Henrique D (2013) Generation and Characterization of a Novel Mouse Embryonic Stem Cell Line with a Dynamic Reporter of Nanog Expression. PLoS ONE 8(3): e59928. doi:10.1371/journal.pone.0059928 Generation and Characterization of a Novel Mouse Embryonic Stem Cell Line with a Dynamic Reporter of Nanog Expression Elsa Abranches 0 Evguenia Bekman 0 Domingos Henrique 0 Qiang Wu, National University of Singapore, Singapore 0 1 Instituto de Medicina Molecular and Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa , Lisboa , Portugal , 2 Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Doca de Pedroucos , Lisbon , Portugal Background: The 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 Findings: We 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/Significance: The 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. - Funding: This work was funded by Fundacao para a Ciencia e a Tecnologia, Portugal (SFRH/BPD/26854/2006 to EA and PTDC/SAU-OBD/100664/2008) (http:// alfa.fct.mctes.pt/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Domingos Henrique is a PLOS ONE Editorial Board member and that this does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Embryonic Stem (ES) cells are characterized by their selfrenewal capacity and pluripotenciality [1,2]. These cells can be derived from the inner cell mass (ICM) of the mammalian blastocyst and can be maintained in vitro under very specific culture conditions ([3,4], reviewed in [5]). Due to their properties, ES cells constitute a promising resource for the next-generation of cellular therapies; however, scientific, technological and ethical questions are still preventing the development of ES cell-based techniques. One of the major bottlenecks has been the lack of a conceptual understanding of the pluripotent state, which has not emerged yet from the systematic molecular characterization of various pluripotent stem cells. Recent work has led to a novel view of pluripotency in ES cells as a self-maintaining and intrinsically-controlled ground state [6,7], regulated by a gene regulatory network (GRN) in which the transcription factors (TFs) Nanog, Oct4 and Sox2 (NOS network) play a central role [79]. 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. However, recent work has shown that both Oct4 and Sox2 can also 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. This scenario emphasizes the precarious and volatile nature of this state and challenges the idea of an intrinsic ability of ES cells to sustain their state based on a dedicated genetic network. The question therefore remains as to which functions do the pluripotency factors play in establishing and maintaining the pluripotent state of ES cells. One feature that distinguishes Nanog from its partners Sox2 and Oct4 is the reported heterogeneous expression of this TF in ES cell cultures (and also in the blastocysts ICM), with some cells showing high levels of Nanog expression while others exhibit reduced levels [14,15]. Furthermore, cells with low or no Nanog expression can evolve into a high-expression state, implying that Nanog levels fluctuate in individual ES cells (contrarily to Oct4 and Sox2) [16,17]. Nanog was initially discovered by virtue of its capacity to oppose differentiation-promoting signals, being essential to maintain ES cells in the absence of LIF/STAT3 signalling [18,19]. This led to the hypothesis that fluctuating levels of Nanog confer different degrees of responsiveness to differentiation signals in individual ES cells, resulting in distinct cellular outputs upon differentiation stimuli [16]. The resulting population heterogeneity might be central to the pluripotent state: on one side, it ensures that there is always a fraction of cells primed to differentiate within a stem cell population, a condition of pluripotency; on the other side, it ensures that other cells are resistant to differentiation cues and keep their pluripotency condition, thereby supporting the selfrenewal property. It is therefore crucial to understand how fluctuations in Nanog levels are generated, and how these fluctuations endow ES cells with various degrees of responsiveness to differentiation cues. To address t (...truncated)