Abstract
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.
Highlights
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 Embryonic Stem Cells (ESCs) into identical undifferentiated daughter cells; pluripotency confers ESCs the ability to produce the majority of cell types upon appropriate determinants
We cultured ESCs in three differentiating culturing media: first, we used regular medium without Leukemia inhibitory factor, a cytokine required for ESC self-renewal; the second culture medium was obtained adding 1% of Dimethylsulfoxide, which is a chemical inducer of ESCs differentiation across all three germ layers [12]; the third culture condition was RM supplemented with Retinoic Acid at 1.5 μM, which is a crucial signaling molecule during embryonic morphogenesis and ESC differentiations [13,14]
We evaluated whether Zscan4 induction reflects an increase of Zscan4 metastate percentage within ESC cultures through RNA in situ hybridization (ISH) (Fig 1B)
Summary
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]. Zscan 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 Zscan metastate is heterogeneously marked by Gm12794 that defines a notcanonical pluripotency signature required for ESCs maintenance [10,11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
