During mammalian development, the fertilized egg (zygote) and its descendent cells up to the eight-cell stage are totipotent — any single cell can differentiate to form all the cells of an organism, and give rise to fetal and placental tissues. This property, however, is lost by the time development has reached the 32-cell blastocyst stage. Therefore, embryonic stem (ES) cells, which are derived from the inner cell mass of blastocysts, are not considered to be totipotent, but pluripotent: they can produce all the cell types of a fetus, but rarely those of the placenta. However, Macfarlan et al.1 (page 57) show that some cells in ES-cell cultures can differentiate into both embryonic and extraembryonic tissues. What's more, most, if not all, ES cells regularly transit this 'totipotent-like' state.

Maternally inherited gene products initially control cellular processes in mammalian zygotes, and it is only at the two-cell stage that the zygote's own genome is activated and assumes full control of development. Interestingly, many of the genes expressed at this stage are retroelements2 — DNA sequences of viral origin that can duplicate themselves and 'jump' into other locations in the genome. Indeed, Macfarlan et al. found that expression of many of the genes activated at the two-cell stage in mouse embryos is driven by regulatory sequences derived from a type of retroelement known as MERVL. Such genes determine the fate of the precursors of placental and fetal cells shortly thereafter (Fig. 1a).

Figure 1: A 'totipotent-like' state in embryonic stem cells.
figure 1

a, During the early development of a mouse embryo, many genes are transiently expressed after the first division of the fertilized egg (zygote). Macfarlan et al.1 find that some of those genes contain virus-derived sequences that drive the genes' transient expression at the two-cell stage. Further cellular divisions result in the development of a blastocyst, a mass of cells that have defined fates: those in the inner cell mass give rise to the fetus, whereas the rest contribute to extraembryonic tissues such as the placenta. b, Embryonic stem (ES) cells, which are derived from the inner cell mass of blastocysts, self-renew in culture (curved arrow). The authors show that most, if not all, ES cells pass through a short-lived state during which they display features that are typical of the totipotent two-cell stage: unlike the rest of the ES cells in the culture, they lack expression of the proteins Oct4, Sox2 and Nanog, and have the ability to form cells of both the placenta and the fetus.

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To label the two-cell stage, the authors introduced into zygotes a synthetic 'reporter' gene that contained a MERVL regulatory sequence and also encoded a red fluorescent protein. As expected, the reporter's expression was intense at the two-cell stage but then gradually faded away and was not observed in blastocysts. However, cultures of ES cells into which the reporter gene had been inserted unexpectedly also contained a few cells (about 0.2–1.5%) that produced the fluorescent protein. Moreover, these rare cells had an overall gene-expression profile that was similar to that of two-cell embryos and was clearly different from that of the rest of the ES cells in the cultures.

Macfarlan et al. found the unusual 'two-cell-like' ES cells under all the culture conditions they tested, although in variable numbers. Most importantly, they showed that most, if not all, ES cells passed through a transient two-cell-like state at some time (Fig. 1b). This result is consistent with previous findings of fluctuating expression of other reporter genes in ES cells3,4,5,6,7,8. Notably, the authors observed cells with similar properties in cultures of induced pluripotent stem cells — such cells are not obtained from blastocysts but by 'reprogramming' differentiated cells such as fibroblasts. Therefore, the presence in ES-cell cultures of a few cells showing two-cell-like gene expression was not due to contamination with cells from early-stage embryos, but is a feature that is probably shared by all pluripotent stem cells.

Crucially, the researchers established that the rare ES cells, but not the rest of the ES cells in a culture, can contribute to both fetal and placental tissues, thereby fulfilling a key attribute of totipotency. However, it remains to be seen whether the two-cell-like ES cells have the potential to generate a complete, live organism.

What is the significance of the two-cell-like ES cells? To address this question, it is important to note that cells from two-cell embryos or early blastocysts represent transient states. Unlike ES cells, they do not self-renew but progress to the next developmental stage. Furthermore, both symmetrical and asymmetrical cell divisions follow the two-cell stage and establish which blastocyst cells will develop into the fetus and which will become extraembryonic tissues9. By contrast, ES cells self-renew through symmetrical divisions. So how can the two-cell-like ES cells generate extraembryonic tissues? A precise analysis of single ES cells is required to understand how they generate different cell types, and whether the cells' fate is determined stochastically or is a pre-programmed property of individual cells. For example, do key molecular determinants of different fetal and placental lineages become segregated into individual cells when they differentiate from two-cell-like ES cells?

The authors found that the two-cell-like cells, unlike the rest of the ES cells, did not produce the proteins Oct4, Sox2 and Nanog, which are typically associated with pluripotency, and instead expressed several genes that are commonly active in two-cell embryos. Notably, one of these genes encodes the protein Zscan4, which is required for the maintenance of telomeres (the ends of chromosomes, which are eroded every time DNA duplicates) and for genomic stability. Lack of Zscan4 leads to a gradual decline in the proliferative capacity of ES cells10. Macfarlan et al. observed that the rare ES cells also displayed a two-cell-like pattern of epigenomic marks — chemical modifications of DNA, and of DNA-bound proteins, that do not alter DNA sequence but affect gene expression.

Therefore, fluctuating patterns of gene expression might provide pluripotent stem cells with a window of opportunity to enter specific cell states. In particular, transition of ES cells through a two-cell-like state may be crucial for 'resetting' the epigenome, for the repair and maintenance of telomeres, and for refreshing the core genetic network underlying pluripotency. Future research, particularly at the single-cell level, may help to reveal why, and how, these cells go through such fluctuating states. It may also advance our knowledge of the mechanisms of cellular rejuvenation and reprogramming in early germ cells, which eventually develop into sperm and eggs. These cells are considered to be immortal, as they have the potential to generate a whole organism and therefore all subsequent generations.