Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

Economou et al. BMC Developmental Biology (2015) 15:35 DOI 10.1186/s12861-015-0084-7 RESEARCH ARTICLE Open Access Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation Constantinos Economou1†, Anestis Tsakiridis1†, Filip J. Wymeersch1, Sabrina Gordon-Keylock1, Robert E Dewhurst2, Dawn Fisher1, Alexander Medvinsky1, Andrew JH Smith1 and Valerie Wilson1* Abstract Background: Pluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. Methods: We manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/ tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively. Results: Here, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo. Conclusions: Our findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency. Keywords: Extragonadal teratoma, Pluripotency, Oct4, Nanog, Brachyury, Inducible expression * Correspondence: † Equal contributors 1 MRC Centre for Regenerative Medicine, School of Biological Sciences, SCRM Building, The University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK Full list of author information is available at the end of the article © 2015 Economou et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Economou et al. BMC Developmental Biology (2015) 15:35 Background In mammals, pluripotency, the ability to generate derivatives of all three embryonic germ layers, is an essential characteristic of cells in the preimplantation and very early postimplantation epiblast as well as their in vitro counterparts, embryonic stem (ES) and Epiblast Stem (EpiSC) cells respectively. The maintenance of a pluripotent state is mediated by the activity of a core transcriptional network dominated by the principal pluripotency factors Oct 4, Nanog and Sox2 (reviewed in [1]. Shortly after the start of gastrulation, although cell fate becomes regionalised [2], epiblast cells remain pluripotent until the beginning of somitogenesis [3]. We have previously shown that ectopic Oct4 expression is the minimal requirement for reinstating pluripotency in normally non-pluripotent somitogenesis-stage embryonic cells explanted in vitro in EpiSC conditions [3]. Furthermore, simultaneous ectopic expression of only four pluripotency factors is sufficient to reprogram embryonic fibroblasts or adult somatic cells to an ES cell-like pluripotent state [4, 5]. A key feature of all pluripotent cells is their capacity to seed teratocarcinomas after engraftment into permissive sites in the adult such as the kidney capsule. Teratocarcinomas comprise differentiated tissues derived from all three germ layers as well as a self-renewing, pluripotent component, embryonal carcinoma (EC) cells [6]. In humans, teratocarcinomas and teratomas, their more benign versions lacking obvious EC cells, are classified as germ cell tumours as, by puberty, they mainly occur in the testis and ovary (for a review see [7]). In neonates, however, the majority of teratomas are extragonadal and occur along the midline, most frequently in the sacrococcygeal region [8, 9]. This may indicate that primordial germ cells (PGCs) initiate these tumours, as they pass along the midline to reach the genital ridges. An alternative hypothesis is that a somatic cell type may acquire pluripotency resulting in teratocarcinogenesis. The midline location of these tumours might in this case indicate either a susceptible somatic cell type or a permissive environment [10, 11]. In support of the latter hypothesis, ectopic Oct4 reactivation in somatic cells from somitogenesis-stage embryos grafted to the adult kidney capsule has been shown to induce teratocarcinoma formation [3]. Furthermore, the transient induction of the main reprogramming factors, Oct4, Sox2, Klf4 and cMyc in vivo drives the emergence of teratomas in a variety of somatic cell types in the adult mouse [12, 13]. Nevertheless, injection of pluripotent cells into a compatible embryonic environment, the blastocyst [14] or postimplantation embryo [15] leads to assimilation of those cells into normal development. Thus the genesis of teratomas/teratocarcinomas depends on the activity of a minimal set of pluripotency factors combined with a permissive environment. Page 2 of 15 The predominant occurrence of e (...truncated)