Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4

RESEARCH ARTICLE Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4 Daniela Tagliaferri1, Maria Teresa De Angelis1, Nicola Antonino Russo1, Maria Marotta1, Michele Ceccarelli2, Luigi Del Vecchio3, Mario De Felice1,3, Geppino Falco1,4,5* 1 Biogem, Istituto di Ricerche Genetiche Gaetano Salvatore Biogem scarl, Ariano Irpino, Italy, 2 Qatar Computing Research Institute, HBKU, Doha, Qatar, 3 Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Naples, Italy, 4 Department of Science, Università degli Studi del Sannio, Benevento, Italy, 5 Department of Biology, University of Naples “Federico II”, Naples, Italy * Abstract OPEN ACCESS Citation: Tagliaferri D, De Angelis MT, Russo NA, Marotta M, Ceccarelli M, Del Vecchio L, et al. (2016) Retinoic Acid Specifically Enhances Embryonic Stem Cell Metastate Marked by Zscan4. PLoS ONE 11(2): e0147683. doi:10.1371/journal.pone.0147683 Editor: Michael Schubert, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, FRANCE Received: September 2, 2015 Accepted: January 7, 2016 Published: February 3, 2016 Copyright: © 2016 Tagliaferri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The microarray data submission was approved and NCBI GEO accession number is GSE75977. Funding: This work was supported by European International Reintegration Grant Marie Curie FP7th; FIRB MIUR and Biogem Research Institute “Gaetano Salvatore”, InterOmics, IEOS, CNR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Pluripotency confers Embryonic Stem Cells (ESCs) the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. Although the majority of ESCs divide without losing pluripotency, it has become evident that ESCs culture consists of multiple cell populations with different degrees of potency that are spontaneously induced in regular ESC culture conditions. Zscan4, a key pluripotency factor, marks ESC subpopulation that is referred to as high-level of pluripotency metastate. Here, we report that in ESC cultures treated with retinoic acid (RA), Zscan4 ESCs metastate is strongly enhanced. In particular, we found that induction of Zscan4 metastate is mediated via RA receptors (RAR-alpha, RAR-beta, and RAR-gamma), and it is dependent on phosphoinositide-3-kinase (PI3K) signaling. Remarkably, Zscan4 metastate induced by RA lacks canonical pluripotency genes Oct3/4 and Nanog but retained both self-renewal and pluripotency capabilities. Finally we demonstrated that the conditional ablation of Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In conclusion, our research provides new insights about the role of RA signaling during ESCs high pluripotency metastate fluctuation. Introduction Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocyst and are characterized by two main peculiarities, namely self-renewal and pluripotency: self-renewal is defined as the symmetrical division of ESCs into identical undifferentiated daughter cells; pluripotency confers ESCs the ability to produce the majority of cell types upon appropriate determinants. It has become evident over the past few years that ESCs fluctuate among different levels of potency as a consequence of paracrine effects and cell-to-cell interactions that are not homogeneously regulated within current in vitro culture conditions [1,2,3]. The ESCs culture PLOS ONE | DOI:10.1371/journal.pone.0147683 February 3, 2016 1 / 15 RA Enhances Zscan4 Metastate heterogeneity may be revealed by the expression of genes that distinctly mark ESCs subpopulations that are commonly defined metastates. Recent evidence reported that the ESCs metastate marked by Zscan4 (zinc finger and SCAN domain containing 4) is characterized by the remarkable potential to produce both embryonic and extra-embryonic cell lineages, therefore it is referred to as totipotent ESCs metastate [4,5,6,7]. The Zscan4 metastate is activated in about 3–5% of the ESC population at any given time and it is required to prevent senescence thus improving the quality of long-term culture. The Zscan4 metastate is enriched in high pluripotency culture conditions by the coordinated actions of extrinsic regulators, signaling pathways, and transcription factors. In particular, the cytokine Leukemia Inhibitory Factor (Lif) is implicated in the establishment of the Zscan4 ESCs metastate, while Phosphoinositide-3-kinase (PI3K) signaling has been reported to regulate Zscan4 expression [8]. Although Zscan4 ESC fluctuation is stabilized by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK) and Glycogen synthase kinase-3 (Gsk-3) signaling inhibition (2i) [9], the Zscan4 metastate is heterogeneously marked by Gm12794 that defines a notcanonical pluripotency signature required for ESCs maintenance [10,11]. In the current study, we investigated how differentiation stimuli such as Dimethylsulfoxide (DMSO), Lif removal, and Retinoic Acid (RA) could affect Zscan4 metastate balance. Interestingly, we found that the Zscan4 metastate was negatively affected by Lif removal and by DMSO treatment, meanwhile it was significantly increased in RA culture condition. We have extended these results by evaluating RA induced Zscan4 metastate through global expression profile, and both self-renewal, and pluripotency capabilities. Our data showed that although the Zscan4 metastate retains self-renewal and pluripotency capabilities, it is also characterized by key markers of ectoderm lineage such as cadherin20 (Cdh20), and brain derived neurotrophic factor (Bdnf). Consistently, the conditional ablation of the Zscan4 subpopulation is dispensable for both endoderm and mesoderm but is required for ectoderm lineage. In our opinion, the Zscan4 metastate enhanced by RA is primed to early ectoderm differentiation and represents a suitable opportunity to characterize key molecular signaling underlying the fluctuation between pluripotency maintenance, and early specification. Materials and Methods Cell culture E14Tg2a.4 ES cells, derived from strain 129P2/OlaHsd were purchased from ATCC company and were cultured for two passages on gelatin-coated feeder-free plates and subsequently maintained in gelatin-coated six-well plates in complete ES medium: GMEM (Glasgow Minimum Essential Medium, Gibco), 15% FBS (EuroClone), 1,000 U ml-1 leukaemia inhibitory factor (LIF) (EuroClone), 1 (...truncated)