Search

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

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.

Embryonic Stem cells (ESCs) can be differentiated into ectoderm, endoderm, and mesoderm derivatives, producing the majority of cell types. In regular culture conditions, ESCs' self-renewal is maintained through molecules that inhibit spontaneous differentiation enabling long-term cellular expansion. This undifferentiating condition is characterized by multiple metastable states that fluctuate between self-renewal and differentiation balance. Here, we aim to characterize the high-pluripotent ESC metastate marked by the expression of Zscan4 through a supervised machine learning framework based on an ensemble of support vector machine (SVM) classifiers. Our study revealed a leukaemia inhibitor factor (Lif) dependent not-canonical pluripotency signature (AF067063, BC061212, Dub1, Eif1a, Gm12794, Gm13871, Gm4340, Gm4850, Tcstv1/3, and Zfp352), that specifically marks Zscan4 ESCs' fluctuation. This novel ESC metastate is enhanced by high-pluripotency culture conditions obtained through Extracellular signal Regulated-Kinase (ERK) and Glycogen synthase kinase-3 (Gsk-3) signaling inhibition (2i). Significantly, we reported that the conditional ablation of the novel ESC metastate marked by the expression of Gm12794 is required for ESCs self-renewal maintenance. In conclusion, we extend the comprehension of ESCs biology through the identification of a novel molecular signature associated to pluripotency programming.

Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on the control genes showed that they behave as expected.