Roadmap Hints at How Stem Cells Reprogram to Earlier Embryonic State

Researchers in Germany and France have studied cells in vitro to identify how changes in gene expression and regulation allow pluripotent embryonic stem (ES) cells to reprogram back to a totipotent-like state found in the earliest two-cell embryos. The scientists, headed by a team at the Helmholtz Zentrum München and Ludwig-Maximilians-Universität München (LMU), hope that their findings may ultimately allow researchers to manipulate ES cells and generate totipotent cells in the lab. They report their findings in Nature Genetics, in a paper entitled “A Molecular Roadmap for the Emergence of Early-Embryonic-Like Cells in Culture.”

Cellular plasticity in multicellular organisms refers to the ability of cells to differentiate into different cell types. Multicellular organisms develop from a single fertilized egg cell, which demonstrates the ultimate plasticity, or totipotency because it has the capacity to generate the complete organism and also the extraembryonic placental tissue. This single fertilized cell divides into two cells—referred to as the 2-cell stage—which also retain totioptency. As cell divison continues, however, the embryonic cells lose totipotency and become pluripotent—they can develop into all the cells of the organism, but not the extraembryonic tissue.

While the state of ES cell pluripotency has been well studied at the gene expression level, “the molecular features of totipotency remain largely unknown,” write Maria-Elena Torres-Padilla, Ph.D.,  and colleagues in the Nature Genetics paper.  Prof. Torres-Padilla is director of the Institute of Epigenetics and Stem Cells (IES) at Helmholtz Zentrum München and professor of stem cell biology at the LMU.

About 1% of cells in a culture of pluripotent ES cells will spontaneously transition back into the totipotent-like state of cells of the two-cell embryo, and are referred to as 2-cell-like cells (2CLCs). Working with colleagues in Germany at the Max Planck Institute for Molecular Biomedicine, and
at the Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS–INSERM) in Strasbourg, France, Torres-Padilla’s team has now uncovered genetic mechanisms involved in the stage-by-stage transformation of pluripotent ES cells to 2CLCs in vitro.

They first compared the genes expressed in ES cells with those expressed in 2CLCs. Studies have indicated that 2CLCs exhibit a transcriptome that is “highly similar” to that of two-cell-stage embryos, including expression of Zscan4 family genes and MERVL. With this knowledge as a starting point, the team tagged ES cells with a marker that would fluoresce when they started expressing MERVL. “MERVL is a retrotransposon expressed in 2CLCs,” explains Diego Rodriguez-Terrones, co-first author. “Using this cell line allows us to separate 2CLCs from the ES cells in the culture by collecting the green cells which have entered the 2-cell-like state. We then compare the genes expressed in both cell types.”

The team then analyzed the transcriptoms of single cells, using computational principal-component analysis, to track the gene expression profiles of individual cells transitioning from an ES cell state to 2CLCs. This allowed them to determine the expression of genes in cells at various states of transition. The results confirmed that 2CLCs arise primarily from precursor cells that express the transcription factor Zscan4. Live cell imaging using a reporter line that expressed a red fluorescent protein when Zscan4 is expressed, also showed that the majority of cells became red (Zscan4-positive) before becoming green (MERVL-positive 2CLCs). “This observation, combined with the transcriptomic data, told us that cells transition through an intermediate state before becoming 2CLCs,” Torres-Padilla notes. “Based on these seemingly ordered changes in gene expression, we wanted to find out what might be driving the emergence of the 2-cell-like state. This information would be crucial for furthering our knowledge concerning key regulators of cellular plasticity.”

Further investigation indicated that Zscan4-expressing cells also exhibit downregulated levels of pluripotency factors, although the authors concluded that the mechanisms by which ES cells exit pluripotency as they transition to a more totipotent state is different from that by which ES cells would lose pluripotency during differentiation. Further analysis of publicly available datasets indicated that chromatin accessibility also changes as cells transition from ES cell to a 2CLC state, with 2CLCs exhibiting an open chromatin structure at MERVL sites, which Zscan4 expressing cells do not.

To try and identify chromatin regulatory elements that promote the transition from ES cell to 2CLC, the authors then applied an small interfering RNA (siRNA) library to knock down expression of 1167 genes and see which had an impact on the emergence of 2CLCs. “The results of this screen were extraordinary, because we identified many novel proteins that regulate the emergence of 2CLCs,” comments Xavier Gaume, Ph.D., co-first author of the paper, who also works at the Torres-Padilla lab.

An analysis of the protein networks for the top 49 validated hits from the screen highlighted five major complexes that appear to regulate the emergence of 2CLCs, “including 23 components of the spliceosome, 4 major members of the EP400–TIP60 (KAT5) complex, 7 members of Polycomb repressor complex 1 (PRC1) and proteins involved in DNA replication,” the authors write. The observation that inhibiting the chromatin factor EP400-Tip60 results in greater numbers of 2CLCs was of particular interest, as EP400-Tip60 is involved in chromatin compaction, which indicates a link between chromatin “openness” and increased cell potency.

“We have identified an intermediate cellular state during the transition to the 2-cell-like state that is characterized by a transcriptional profile distinctive from those of ES and 2-cell-like cells,” the authors conclude. “Our data also indicate that 2-cell-like cells themselves are heterogeneous, but the nature of their heterogeneity differs from that of ES cells.”