Previous studies have shown that embryonic stem cell (ESC) cultures are heterogeneous with respect to certain transcription factors (e.g. Dppa3, Nanog, Sox17 and Gata6) and could imbue specific cells with distinct attributes (Hayashi et al and Singh et al). Now in a study in Nature, researchers from the group of Samuel L. Pfaff at The Salk Institute for Biological Studies, California have identified a transient cell population in both ESC and induced pluripotent stem cell (iPSC) cultures that expresses genes usually observed in the totipotent 2-cell embryo (Macfarlan et al). The group go on to demonstrate that this expression profile is driven by regulatory regions of endogenous retroviruses, which are known to be actively expressed at the 2-cell stage (Evsikov et al, Kigami et al and Peatson et al) but repressed thereafter (Ribet et al and Svoboda et al), and that this regulatory mechanism may have helped to drive cell-fate regulation in placental mammals (Macfarlan et al).

Initial RNA-sequencing of the 2-cell mouse embryo demonstrated that the most highly activated repeat was the MuERV-L (murine endogenous retrovirus with leucine tRNA primer) family of retroviruses and their corresponding long terminal repeat (LTR) promoters. Expression of the promoter regions led to the generation of 626 chimaeric transcripts, with 90% being 5’ LTR–exon fusions that generated open reading frames (ORFs), suggesting that these LTRs had become functional promoters for protein-coding genes. Developmental regulation of expression of these promoters was shown through the appearance of the retrovirally-encoded Gag protein and the lack of Oct4 protein at the 2-cell stage and the loss of Gag and gain of Oct4 expression by the blastula stage.

Retroviral activity was then analysed using the presence of Gag and a construct in which the red fluorescent protein tandem dimeric Tomato (tdTomato) is driven by MuERV-L 5’ LTR activity.   Interestingly, this led to the discovery of rare cells that expressed Gag and tdTomato alongside MuERV-L mRNA in cultures of transfected ESCs.   Sorted tdTomato⁺ ESCs expressed much higher levels of MuERV-L transcripts than the tdTomato^- population, while tdTomato⁺ ESCs were also enriched for genes associated with the 2-cell stage (Zscan4, Tcstv1/3, Eif1a, Gm4340 (Thoc4), Tdpoz1–5 and Zfp352) including 52 genes that generated chimaeric transcripts linked to MuERV-L elements.   Interestingly, the tdTomato⁺ ESCs did not express the pluripotency markers Oct4, Sox2 and Nanog at the protein level although their mRNA levels were unaltered. Proof that these cells did not arise from contamination with trophectoderm or primitive endoderm came via the generation of iPSCs from mouse fibroblasts and the subsequent identification of cells expressing Gag but with no Oct4 protein, similar to ESCs. Next, using a complex genetic labelling scheme to indelibly mark ESCs that expressed 2-cell genes, it was found that nearly every cell within a culture passed through the 2-cell-like stage, and through cell sorting it was further shown that that  tdTomato⁺ cells could produce tdTomato^- cells and vice versa, suggesting the reversible transient state of this cellular phenotype.

Further analysis of the 2-cell state found that tdTomato⁺ cells had higher levels of permissive histone marks (methylation of lysine 4 histone H3 and acetylation of histones H3 and H4), similar to that observed in 2-cell embryos (Wiekowski et al) while DNA methylation analysis demonstrated hypomethylation of MuERV-L sequences in tdTomato⁺ cells compared with tdTomato^- cells, again indicative of a transcriptionally permissive state. The importance of the chromatin environment to the 2-cell state was again demonstrated by the upregulation of MuERV-L and several 2-cell genes in ESCs with mutated Kdm1a (histone H3 Lysine 4 and 9 demethylase), Kap1 (KRAB -associated transcriptional repressor Kap1) or G9a (histone H3 lysine 9 methyltransferase) or with histone deacetylase inhibitor (trichostatin A) treatment.

The totipotentiality of the 2 cell-like ESCs was next assayed through the injection of tdTomato⁺ and tdTomato^- ESCs into morula-stage embryos. While the tdTomato^- ESCs only contributed to the inner cell mass (ICM) of the resultant blastocyst, the tdTomato⁺ ESCs contributed to the trophectoderm in addition to the ICM, suggesting their expanded cell fate potential. Later development was studied through infection of tdTomato ESCs with a constrictively active GFP construct, and demonstrated that tdTomato⁺GFP⁺ ESCs contributed to embryonic endoderm, ectoderm, mesoderm, the germ lineage as well as the yolk sac and placenta. Interestingly, Kdm1a homozygous mutant ESCs also contributed to embryonic tissues, the amnion, yolk sac and placental tissues, including giant trophoblast cells and primordial germ cells, suggesting that the loss of this chromatin modifying enzyme is sufficient to expand cell fate potential in ESCs. Lastly, the depletion of tdTomato⁺ cells from ESC cultures was analysed, through a scheme which allowed for genetic ablation with diptheria toxin (DTA). After 20 passages, cultures depleted of 2-cell state cells could still contribute to high grade chimaeras although their differentiation was biased towards mesoderm and ectodermal lineages in vitro, suggesting that the  2C-like state may aid in the preservation of the broad fate potential of ESC.

Overall, this exciting piece of data suggests that retrotransposons may have played important roles in mammalian development and potency by contributing to the specification of cell types and leading to the formation of placental tissues, supported by the observations that endogenous retroviruses are found in all placental mammals (Benit et al) and that they have been shown to have had a crucial role in the “rewiring” of gene regulatory networks in ESCs (Kunarso et al).Further work will be required to establish how MuERV-L and associated 2-cell stage genes regulate the observed expanded cell fate, but exploration of the practical uses of these cells may be useful with regards to differentiation studies with an eye on clinical usefulness.

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STEM CELLS correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.

Original study from Nature.