Scientists from the Helmholtz Zentrum München (German Research Center for Environmental Health) identified a specific population of pluripotent embryonic stem cells that can reprogram into totipotent-like cells in culture. Moreover, researchers, who published an article with the results on December 18, 2017 in Nature Genetics, determined the factors and mechanism of this reprogramming.
Cellular plasticity is the ability to create different types of cells. After fertilization of the egg with the sperm, a cell (zygote) is formed, which at early stages of development has a high plasticity – all types of cells of the body are formed from it. Therefore, cellular plasticity is necessary for the existence of complex multicellular organisms, such as humans.
Zygote in the first stage of crushing divides into two cells, which are called blastomeres. These cells have the highest level of cellular plasticity: they are totipotent – this means that they can create a full-fledged organism, including the extra-embryonic placental tissue. Embryonic stem cells are pluripotent – they can produce all cells of the body, with the exception of extraembryonic cells.
In a culture of embryonic stem cell (ESC), a small population (about 1%) spontaneously turns into cells similar to totipotent embryonic cells in the two-cell stage of the zygote fragmentation. Such cells are called 2-cell-like cells (2CLCs).
In this study, Prof. Dr. Maria Elena Torres-Padilla’s team set out to determine the specific molecular nature of these cells and find out how they come to be.
The aim of the team was to gain insights in to the molecular features of totipotency and to work out the process of changing the plasticity of cells. The ultimate goal of the group was to study the behavior of these totipotent-like cells in order to be able to manipulate them and create them in vitro.
The team began by comparing the genes expressed in ES cells to those expressed in 2CLCs. To do this, they used embryonic stem cells which express a green fluorescent protein when the cells begin to express the MERVL gene.
“MERVL is a retrotransposon expressed in 2-cell-like cells”, – explains Diego Rodriguez-Terrones, a PhD student in the Torres-Padilla lab and co-first author of the paper. “Using this cell line allows us to separate 2-cell-like cells 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”, – he adds
This single cell transcriptome analysis followed by computational analyses allowed the scientists to identify gene expression profiles of cells during the transformation from ES cells to 2CLCs.
The team found that during the transition period, the cells expressed an increasing amount of the gene encoding the transcription factor Zscan4.
The researchers developed their reporter line to also be able to express a red fluorescent protein when Zscan4 is expressed. The visualization of living cells confirmed that most of cells became red (it became Zscan4-positive) before becoming green (MERVL-positive 2-cell-like cells).
“This observation, combined with the transcriptomic data, told us that cells transition through an intermediate state before becoming 2-cell-like cells”, – said Maria Elena Torres-Padilla.
“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.”
To determine chromatin regulators that can promote cell reprogramming, the team performed screening for siRNA with impaired expression of more than 1000 genes to see how this would affect the onset of 2CLC.
“The results of this screen were extraordinary, because we identified many novel proteins that regulate the emergence of 2CLCs”, – said Dr. Xavier Gaume, co-first author of the paper.
Of particular interest is the fact that lowering the levels of a specific chromatin factor (Ep400/Tip60) results in more 2CLC. Since Ep400/Tip60 participates in chromatin compaction, this observation reveals an interesting link between chromatin “openness” and an increase potency.